Magnetically immobilized biocidal enzymes and biocidal chemicals

ABSTRACT

The present invention provides compositions and methods for reducing microbial and nematodal contamination or infection in plants, animals, fabrics, and products therefrom. The present invention also provides compositions and methods for reducing human infections and the emergence of antimicrobial resistance. In particular, the invention provides magnetic nanoparticles comprising biocidal or biostatic enzymes in one component, substrates for the enzymes in a second component, and a biocidal chemical agent that works in combination or synergistically with the enzymes. The compositions are dormant and become active upon exposure to hydration, oxygen, or mixing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/374,836, filed Aug. 13, 2016, and U.S. Provisional Application No.62/511,331, filed May 25, 2017, both of which are incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for reducingmicrobial and nematodal contamination or infection in plants, animals,fabrics, and products therefrom. The present invention also providescompositions and methods for reducing human infections and the emergenceof antimicrobial resistance. In particular, the invention providesmagnetic nanoparticles comprising biocidal or biostatic enzymes in onecomponent, substrates for the enzymes in a second component, and abiocidal chemical agent that works in combination or synergisticallywith the enzymes. The compositions are dormant and become active uponexposure to hydration, oxygen, or mixing.

BACKGROUND OF THE INVENTION

Contaminating and infectious microorganisms significantly reduce theyield, quality, and safety of agricultural and animal productsworldwide. The resulting economic losses are in the hundreds of billionsof dollars annually in the United States alone. In addition, currentmethods for reducing plant and animal infections rely heavily on the useof antimicrobial chemicals that may result in fungicide and antibioticresistance that, in turn, increases the probability of selecting fordrug-resistant plant, animal, and human pathogens. These microbes havebeen selected to survive in the presence of medically and agriculturallyimportant antimicrobial chemicals and are a significant threat to humanhealth and food security.

For example, Antibiotic Resistant Microbes (ARM) are a growing publichealth concern because infections have become increasingly difficult andexpensive to treat. Concern turns into crisis in hospital environments.Antibiotics of last resort such as vancomycin, are steadily becomingineffectual against superstrains. Carbapenem ResistantEnterobacteriaceae (CRE), some of the most ubiquitous microbes in theenvironment, are now resistant to almost all antibiotics. CRE infectionsare so difficult to treat that 50% of patients infected by them die. Inthe 2013 Antibiotic Resistance Threat report, the CDC identifies threemajor concerns: 1) new active molecules are harder to discover andproduce, 2) development costs are prohibitive, and 3) resistance spreadsfaster than ever. The World Health Organization (WHO) warns that “[i]nthe absence of urgent corrective and protective actions, the world isheading toward a post-antibiotic era, in which many common infectionswill no longer have a cure and [will], once again, kill unabated.”(World Health Day, Combat Drug Resistance: No Action Today Means No CureTomorrow, Statement by WHO Director-General, Dr. Margaret Chan, Apr. 6,2011,http://www.who.int/mediacentre/news/statements/2011/whd_20110407/en/.)In 2013, the Center for Disease Control (CDC) estimated that 70 percentof the bacteria that caused hospital-acquired infections were resistantto at least one of the relevant antibiotics. (Antibiotic ResistanceThreats in the United States, 2013, Centers for Disease Control andPrevention: Atlanta, Ga.,http://www.cdc.gov/drugresistance/threat-report-2013/.) It has long beenargued by public advocacy groups, such as the Alliance for the PrudentUse of Antibiotics, that antibiotics are societal drugs. Individual useaffects the entire community.

Such antibiotic resistance has become a worldwide concern now that theconsequences of antibiotic overuse are being studied and reported. TheCDC now estimates that in the U.S. at least 23,000 people die frommultiple antibiotic-resistant bacteria infections every year. Id. In theU.S. alone, these superbug infections are responsible for $20 billion inexcess healthcare costs, $35 billion in societal costs, and 8 millionadditional hospital stays each year. (Roberts et al., Clin. Infect. Dis.49(8):1175-84 (2009).)

In agriculture, seeds can spread plant bacterial and fungal diseasesacross farms, states, and countries. In some instances, seedlings faildue to “damping off” This is the death of a seedling before or shortlyafter emergence due to decomposition of the root and/or lower stem.Control of such diseases may begin with the seeds. Seed treatmentsshould protect seeds from pathogens such as bacteria, viruses, andfungi. Thus, high-quality, disease-free seeds are an important part ofobtaining higher plant yields and food safety.

In agriculture, bacteria can also contaminate animal environments inhigh density breeding operations. Pathogenic microbes such as Salmonellaand Listeria entering the food chain cost hundreds of millions ofdollars in product recalls and hospitalizations. (Hoffmann, S. and T. D.Anekwe, Making Sense of Recent Cost-of-Foodborne-Illness Estimates,United States Department of Agriculture, Economic Research Service,2013,http://www.ers.usda.gov/publications/eib-economic-information-bulletin/eib118.aspx;Hoffmann et al., J. Food Prot. 75(7): 1292-1302 (2012).)

Some food borne pathogens are also known to be becomeantibiotic-resistant. Foodborne pathogens have a vast impact onAmericans, causing 48 million illnesses, 28,000 hospitalizations, and atleast 3,000 deaths each year.(http://www.cdc.gov/foodborneburden/2011-foodborne-estimates.html.) Theyalso have a tremendous impact on businesses and the healthcare systemresulting in annual costs of $14-$16 billion. This includes directmedical costs and value of time lost to illness. Bacteria are theprimary culprits. They comprise four of the top five pathogens thatcontribute to illness, three of the top five that causehospitalizations, and three of the top five that cause mortality.

Nematodes are microscopic worms that cause eighty billion dollars ofcrop loss in the world each year. Plant-parasitic nematodes threatencrops throughout the world. In fact, all crops are damaged by at leastone species of nematode. They attack almost every part of the plantincluding roots, stems, leaves, fruits and seeds.

Some 5,000 species of nematodes are estimated to be parasites ofvertebrate animals and humans. These species are often characterized ina larger group of worm parasites as helminths. Strategies for managingnematode parasites of domestic vertebrate animals include control ofsecondary hosts or vectors and the use of chemical anthelminthics.Roundworms can infect dogs, cats, cattle, sheep, pigs, and poultry.

Most parasitic roundworms have direct life cycles, i.e. the free-livingstages that do not need an intermediate host for development. They candirectly infect their final host where they migrate to theirpredilection sites and complete development to adults. Inside the finalhost, pregnant females produce thousands of eggs that are usuallyexcreted with the feces of the host and contaminate pastures, rivers,lakes, etc.

Controlling plant pathogens relies heavily on synthetic chemicals tomaintain high product yields. The public has shown increasing concern,however, over the effects that agrochemical residues have on humanhealth and the environment. (Mark et al., FEMS Microbiol. Ecol.56(2):167-77 (2006); Ritter et al., J. Tox. Environ. Health 9(6):441-56(2006).) Farmers who use synthetic agrochemicals have more neurologicalproblems that include headaches, fatigue, insomnia, dizziness and handtremors. (http://www.niehs.nih.gov/health/topics/agents/pesticides/).Agrochemicals may also cause birth defects, nerve damage, cancer,decreased sperm motility and acute poisoning (Moses, AAOHN J.,37(3):115-30 (1989); Reeves and Schafer Int'l J., Occup. Environ. Health9(1):30-39 (2003); Carozza et al., Environ. Health Perspect.116(4):559-65 (2008); U.S. Environmental Protection Agency, 2014,http://www.epa.gov/pesticides/food/risks.htm). Furthermore, protectingcrops from fungal pathogens is particularly challenging for organiccrops on which synthetic antifungal chemicals cannot be used.

Fungicides and antibiotics are widely used in developed agriculturalsystems to control disease and safeguard crop yield and quality. Overtime, however, resistance to many of the most effective fungicides andantibiotics has emerged and spread in pathogen populations (Lucas etal., Adv Appl Microbiol., 90:29-92 (2015)). The widespread practice ofroutinely dosing farm animals with antifungals and antibiotics iscontributing to this threat. Much of this use is for preventing, ratherthan treating, disease. Drug-resistant microbes carried by farm animalscan spread to humans through consumption of contaminated food, fromdirect contact with animals, or by environmental spread, for example, incontaminated water or soil. Antibiotic and fungicide resistant pathogensof humans and farm animals are emerging and spreading at a rate that maynot be contained by the development of new drugs.

Thus there is a significant need for new methods of controlling fungal,bacterial, oomycete, and nematode pathogens that cause agriculturalcontamination or infections as well as human infections.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for reducingmicrobial contamination or infection in plants, animals, fabrics, andproducts therefrom. The present invention also provides compositions andmethods for reducing human infections. The present invention alsoreduces the emergence of resistance in microbes towards chemicalbiocides. In particular, the invention provides magnetic nanoparticlescomprising microbiocidal and/or microbiostatic enzymes in one component,substrates for the enzymes in a second component, and a chemicalmicrobiocidal agent that works synergistically with the enzymes. Thecompositions are dormant and become active upon exposure to hydration,oxygen, or mixing.

Thus, the invention provides a solid fungicidal composition, comprising;a first component having self-assembled mesoporous aggregates ofmagnetic nanoparticles comprising a hydrogen peroxide producing enzymeand a free radical producing enzyme; a second component having a firstsubstrate for said hydrogen peroxide producing enzyme and a secondsubstrate for said free radical producing enzyme; and a chemicalfungicide; wherein said composition is essentially inactive, whereinexposure of said first and second components to hydration or oxygenactivates said composition and results in said substrate for saidhydrogen peroxide producing enzyme being oxidized into hydrogenperoxide, wherein said hydrogen peroxide acts as a substrate for saidfree radical producing enzyme, and wherein said free radicals areproduced having fungicidal activities.

In some embodiments, said chemical fungicide is selected from the groupconsisting of mefenoxam, myclobutanil, chlorothalonil, prothioconazole,trifloxystrobin, propiconazole, mancozeb, and copper. In a preferredembodiment, said chemical fungicide is chlorothalonil. In anotherpreferred embodiment, said chemical fungicide is mancozeb.

The invention provides a solid bactericidal composition, comprising; afirst component having self-assembled mesoporous aggregates of magneticnanoparticles comprising a hydrogen peroxide producing enzyme and a freeradical producing enzyme; a second component having a first substratefor said hydrogen peroxide producing enzyme and a second substrate forsaid free radical producing enzyme; and a chemical antibiotic; whereinsaid composition is essentially inactive, wherein exposure of said firstand second components to hydration or oxygen activates said compositionand results in said substrate for said hydrogen peroxide producingenzyme being oxidized into hydrogen peroxide, wherein said hydrogenperoxide acts as a substrate for said free radical producing enzyme, andwherein said free radicals are produced having bactericidal activities.

In some embodiments, said chemical antibiotic is selected from the groupconsisting of ampicillin, streptomycin, vancomycin, and copper.

The invention provides a liquid fungicidal composition, comprising; afirst component having self-assembled mesoporous aggregates of magneticnanoparticles comprising a free radical producing enzyme; a secondcomponent having a substrate for said free radical producing enzyme anda hydrogen peroxide source; and a chemical fungicide; wherein saidcomposition is essentially inactive, wherein mixing said first andsecond components activates said composition and results in saidhydrogen peroxide source acting as a substrate for said free radicalproducing enzyme, and wherein said free radicals are produced havingfungicidal activities.

In some embodiments, said chemical fungicide is selected from the groupconsisting of mefenoxam, myclobutanil, chlorothalonil, prothioconazole,trifloxystrobin, propiconazole, mancozeb, an essential oil, and copper.In a preferred embodiment, said chemical fungicide is chlorothalonil. Inanother preferred embodiment, said essential oil is tea tree oil (TTO).

The invention provides a liquid bactericidal composition, comprising; afirst component having self-assembled mesoporous aggregates of magneticnanoparticles comprising a free radical producing enzyme; a secondcomponent having a substrate for said free radical producing enzyme anda hydrogen peroxide source; and a chemical antibiotic; wherein saidcomposition is essentially inactive, wherein mixing said first andsecond components activates said composition and results in saidhydrogen peroxide source acting as a substrate for said free radicalproducing enzyme, and wherein said free radicals are produced havingbactericidal activities.

In some embodiments, the final chemical fungicide concentration isbetween about 10% and 2500% of the half maximal effective concentration(EC₅₀). In other embodiments, the final chemical antibioticconcentration is between about 1 and 100% of the minimum inhibitoryconcentration (MIC) or minimum bactericidal concentration (MBC).

In other embodiments of the invention, the compositions and methodsdisclosed herein comprise microbiocidal compositions that comprise botha chemical antibiotic and a chemical fungicide.

In some embodiments of the invention, said mesoporous aggregates ofmagnetic nanoparticles have an iron oxide composition. In otherembodiments of the invention, said mesoporous aggregates of magneticnanoparticles have a magnetic nanoparticle size distribution in which atleast 90% of magnetic nanoparticles have a size of at least about 3 nmand up to about 30 nm, and an aggregated particle size distribution inwhich at least about 90% of said mesoporous aggregates of magneticnanoparticles have a size of at least about 10 nm and up to 500 nm. Inother embodiments of the invention, said mesoporous aggregates ofmagnetic nanoparticles possess a saturated magnetization of at least 10emu/g.

In some embodiments of the invention, said free-radical-producing enzymeand hydrogen peroxide producing enzyme are contained in said mesoporousaggregates of magnetic nanoparticles in up to about 100% of saturationcapacity.

In some embodiments of the invention, said hydrogen peroxide generatingenzyme is an oxidase. In other embodiments of the invention, saidoxidase is glucose oxidase or alcohol oxidase.

The invention provides an agricultural product comprising the fungicidaland bactericidal compositions disclosed herein. In some embodiments, theinvention provides a liquid pesticide product comprising the fungicidaland bactericidal compositions disclosed herein. In other embodiments,the invention provides a seed coating, comprising the fungicidal orbactericidal compositions disclosed herein. In some embodiments, theinvention provides a seed comprising the seed coatings disclosed herein,wherein said seed is selected from the group consisting of vegetable,fruit, flower and field crop.

In preferred embodiments, said vegetable seed is selected from the groupconsisting of tomato, pea, onion, garlic, parsley, oregano, basil,cilantro, carrot, cabbage, corn, cucumber, radish, pepper, broccoli,cauliflower, cucumber, spinach, kale, chard, artichoke, and lettuce. Inother preferred embodiments, said fruit seed is selected from the groupconsisting of citrus, tomato, orange, lemon, lime, avocado, clementine,apple, persimmon, pear, peach, nectarine, berry, strawberry, raspberry,grape, blueberry, blackberry, cherry, apricot, gourds, squash, zucchini,eggplant, pumpkin, coconut, guava, mango, papaya, melon, honeydew,cantaloupe, watermelon, banana, plantain, pineapple, quince, sorbus,loquata, plum, currant, pomegranate, fig, olive, fruit pit, a nut,peanut, almond, cashew, hazelnut, brazil nut, pistachio, and macadamia.In other preferred embodiments, said field crop is selected from thegroup consisting of corn, wheat, soybean, canola, sorghum, potato, sweetpotato, yam, lentils, beans, cassava, coffee, hay, buckwheat, oat,barley, rape, switchgrass, elephant grass, beet, sugarcane, and rice. Inother preferred embodiments, said flower seed is selected from the groupconsisting of annual, perennial, bulb, flowering woody stem, carnation,rose, tulip, poppy, snapdragon, lily, mum, iris, alstroemeria, pom,fuji, and bird of paradise.

The invention provides an animal bedding, comprising the fungicidal orbactericidal compositions disclosed herein.

The invention provides a wound dressing, comprising the fungicidal orbactericidal compositions disclosed herein.

The invention provides a fabric, comprising the fungicidal orbactericidal compositions disclosed herein.

The invention provides a method of improving a plant product yield,comprising exposing the improved seeds disclosed herein to hydration andoxygenation prior to or during the planting or germination of saidplant.

The invention provides a method of improving an animal product yield,comprising exposing the improved animal bedding disclosed herein tohydration and oxygen prior to or during use by said animal. In apreferred embodiment, said hydration is from said animal's urine. Inother preferred embodiments, said animal product is selected from thegroup consisting of live animals, milk, meat, fat, eggs, bodily fluids,blood, serum, antibodies, enzymes, rennet, bone, animal byproducts, andanimal waste.

In other preferred embodiments, said animal is selected from the groupconsisting of cows, pigs, chickens, turkeys, horses, sheep, goats,donkeys, mules, ducks, geese, buffalo, camels, yaks, llama, alpacas,mice, rats, dogs, cats, hamsters, guinea pigs, reptiles, amphibians,parrots, parakeets, cockatiels, canaries, pigeons, doves, and insects.

The invention provides a method of reducing sepsis, comprisingadministering the improved wound dressings disclosed herein to a wound.

The invention provides a method of producing the fungicidal orbactericidal compositions disclosed herein, comprising formulating saidfirst component with a matrix material selected from the groupconsisting of water-soluble cellulose derivatives, water-solvatablecellulose derivatives, alginate derivatives, and chitosan derivativesand formulating said second component with a matrix material selectedfrom the group consisting of water-soluble cellulose derivatives,water-solvatable cellulose derivatives, alginate derivatives, andchitosan derivatives. In preferred embodiments, said first component isfurther subjected to spray drying, freeze drying, drum drying, pulsecombustion drying, or rotary seed coating. In other preferredembodiments, said second component is further subjected to spray drying,freeze drying, drum drying, pulse combustion drying, or rotary seedcoating.

The invention provides a method of reducing or eliminating fungal orbacterial growth, comprising spraying a substance with the liquidfungicidal or bactericidal compositions disclosed herein.

The invention provides a method of protecting an agricultural productfrom a pathogen, comprising exposing said product to the fungicidal orbactericidal compositions disclosed herein. In preferred embodiments,said pathogen is a plant, animal, or human pathogen. In other preferredembodiments, said pathogen is a fungus, oomycete, or bacterium. In morepreferred embodiments, said fungus is selected from the group consistingof Rhizoctonia species and Fusarium species. In other preferredembodiments, said pathogen is a bacterium selected from the groupconsisting of Xanthomonas campestris, Clavibacter michiganensis,Acidovorax avenae, Pseudomonas viridiflava, Pseudomonas syringae,Escherichia coli, Salmonella species, and Listeria species. In otherpreferred embodiments, said pathogen is an oomycete selected from thegroup consisting of Pythium species and Phytophthora species

The invention provides a method of reducing or eliminating damping offin a plant, comprising exposing said plant to the fungicidal orbactericidal compositions disclosed herein.

The invention provides a solid nematocidal composition, comprising afirst component having self-assembled mesoporous aggregates of magneticnanoparticles comprising a hydrogen peroxide producing enzyme and a freeradical producing enzyme and a second component having a first substratefor said hydrogen peroxide producing enzyme and a second substrate forsaid free radical producing enzyme; wherein said composition isessentially inactive, wherein exposure of said first and secondcomponents to hydration or oxygen activates said composition and resultsin said substrate for said hydrogen peroxide producing enzyme beingoxidized into hydrogen peroxide, wherein said hydrogen peroxide acts asa substrate for said free radical producing enzyme, and wherein saidfree radicals are produced having fungicidal activities.

The invention provides a liquid nematocidal composition, comprising afirst component having self-assembled mesoporous aggregates of magneticnanoparticles comprising a free radical producing enzyme and a secondcomponent having a substrate for said free radical producing enzyme anda hydrogen peroxide source; wherein said composition is essentiallyinactive, wherein mixing said first and second components activates saidcomposition and results in said hydrogen peroxide source acting as asubstrate for said free radical producing enzyme, and wherein said freeradicals are produced having fungicidal activities.

In a preferred embodiment, the nematocidal compositions furthercomprising an essential oil. In more preferred embodiments, saidessential oil is selected from the group consisting of tea tree (TTO),aegle, ageratum, citrus, citronella, orange, pine, eucalyptus, marigold,geranium, lemongrass, orange, palmarosa, mint, peppermint, cinnamon,clove, rosemary, thyme, garlic, oregano, anise, cumin, turmeric,curcuma, caraway, fennel, onion, and patchouli oil. In a most preferredembodiment, said essential oil is TTO.

The invention provides an agricultural product, comprising thenematocidal compositions described herein. In other embodiments, theinvention provides a liquid pesticide product comprising the nematocidalcompositions described herein.

The invention provides a seed coating comprising the nematocidalcompositions described herein. In some embodiments, said seed isselected from the group consisting of vegetable, fruit, flower and fieldcrop. In preferred embodiments, said vegetable seed is selected from thegroup consisting of tomato, pea, onion, garlic, parsley, oregano, basil,cilantro, carrot, cabbage, corn, cucumber, radish, pepper, broccoli,cauliflower, cucumber, spinach, kale, chard, artichoke, and lettuce.

In other preferred embodiments, said fruit seed is selected from thegroup consisting of citrus, tomato, orange, lemon, lime, avocado,clementine, apple, persimmon, pear, peach, nectarine, berry, strawberry,raspberry, grape, blueberry, blackberry, cherry, apricot, gourds,squash, zucchini, eggplant, pumpkin, coconut, guava, mango, papaya,melon, honeydew, cantaloupe, watermelon, banana, plantain, pineapple,quince, sorbus, loquata, plum, currant, pomegranate, fig, olive, fruitpit, a nut, peanut, almond, cashew, hazelnut, brazil nut, pistachio, andmacadamia.

In other preferred embodiments, said field crop is selected from thegroup consisting of corn, wheat, soybean, canola, sorghum, potato, sweetpotato, yam, lentils, beans, cassava, coffee, hay, buckwheat, oat,barley, rape, switchgrass, elephant grass, beet, sugarcane, and rice.

In other preferred embodiments, said flower seed is selected from thegroup consisting of annual, perennial, bulb, flowering woody stem,carnation, rose, tulip, poppy, snapdragon, lily, mum, iris,alstroemeria, pom, fuji, and bird of paradise.

The invention provides an animal bedding, comprising the nematocidalcomposition described herein.

The invention provides a method of improving a plant product yield,comprising exposing the seed of described herein to hydration andoxygenation prior to or during the planting or germination of saidplant.

The invention provides a method of improving an animal product yield,comprising exposing the animal bedding described herein to hydration andoxygen prior to or during use by said animal. In some embodiments, saidhydration is from said animal's urine. In other embodiments, said animalproduct is selected from the group consisting of live animals, milk,meat, fat, eggs, bodily fluids, blood, serum, antibodies, enzymes,rennet, bone, animal byproducts, and animal waste. In other embodiments,said animal is selected from the group consisting of cows, pigs,chickens, turkeys, horses, sheep, goats, donkeys, mules, ducks, geese,buffalo, camels, yaks, llama, alpacas, mice, rats, dogs, cats, hamsters,guinea pigs, reptiles, amphibians, parrots, parakeets, cockatiels,canaries, pigeons, doves, and insects.

The invention provides a method of producing the nematocidal compositiondescribed herein, comprising formulating said first component with amatrix material selected from the group consisting of water-solublecellulose derivatives, water-solvatable cellulose derivatives, alginatederivatives, and chitosan derivatives and formulating said secondcomponent with a matrix material selected from the group consisting ofwater-soluble cellulose derivatives, water-solvatable cellulosederivatives, alginate derivatives, and chitosan derivatives. In someembodiments, said first component is further subjected to spray drying,freeze drying, drum drying, pulse combustion drying, or rotary seedcoating. In other embodiments, said second component is furthersubjected to spray drying, freeze drying, drum drying, pulse combustiondrying, or rotary seed coating.

The invention provides a method of reducing or eliminating nematodegrowth, comprising spraying a substance with the liquid nematocidalcompositions described herein.

The invention provides a method of protecting an agricultural productfrom a nematode, comprising exposing said product to the nematocidalcompositions disclosed herein. In some embodiments, said nematode isselected from the group consisting of Meloidogyne species (spp.),Heterodera spp., Globodera spp., Pratylenchus spp., Helicotylenchusspp., Radopholus similis, Ditylenchus dipsaci, Rotylenchulus reniformis,Xiphinema spp, Aphelenchoides spp., Toxocara spp., Bursaphelenchusxylophilus, and trichinella spiralis. In preferred embodiments, saidnematode is a Meloidogyne spp., a trichinella spiralis, or a Toxocaraspp.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C. Effect of LP:GOx enzyme ratio and substrateconcentration on dry enzyme disk bactericidal efficacy. The analysis wasdone twice and results were consistent across the two replicates. Datafor a single analytical run is shown. The effect of the LP:GOx ratio onzones of inhibition (ZOI) of Xanthomonas campestris pv. vitians (Xcv) isshown at 1× (FIG. 1A) and 10× (FIG. 1B) substrate concentrations. Eachbar represents the mean of two technical replicates. FIG. 1C shows theeffect of 1× vs. 10× substrate concentrations averaged across all fiveLP:GOx enzyme ratios. Each bar represents the mean of ten replicates.Statistical analysis was done using Tukey's Honest SignificantDifference (HSD), P<0.05).

FIG. 2. Effect of LP:GOx enzyme concentration on dry enzyme diskefficacy against fungal growth (Rhizoctonia solani and Fusariumgraminearum). Each bar represents the mean of two replicates. Columnswith an asterisk above them are significantly different (Tukey's HSD,P<0.05).

FIG. 3. Effect of three LP:GOx enzyme concentrations on growth ofPythium ultimum. Plate A is a control with dry substrate disk only.Plate B shows that the lowest enzyme concentration (0.0034× or 0.8 nMenzyme) did not inhibit growth of P. ultimum relative to the control.Plate C shows a medium enzyme concentration (0.68× or 161 nM enzyme)completely inhibited P. ultimum growth. Plate D shows a high enzymeconcentration (2× or 476 nM enzyme) also completely inhibited P. ultimumgrowth.

FIG. 4. Reduction in P. ultimum growth relative to the untreated controlfollowing treatment by Daconil®+enzyme disk (black bars), Daconil® only(gray bars), and enzyme disk only (dotted line). Enzymes concentrationsin enzyme disks were 81 nM.

FIG. 5. Reduction in P. aphanidermatum growth relative to the untreatedcontrol following treatment by Daconil®+enzyme disk (black bars),Daconil® only (gray bars), and enzyme disk only (dotted line). Enzymesconcentrations in enzyme disks were 81 nM.

FIG. 6. Reduction in P. ultimum growth following treatment by Daconil®,enzyme disk, and Daconil®+enzyme disk. Enzyme concentrations in enzymedisks were 81 nM. Smaller colony diameters indicate greater oomycetegrowth inhibition. Plate A shows that a Daconil®-only treatment (0.0078%Daconil®) resulted in an 18% reduction in growth relative to thenon-treated control. Plate B shows a non-treated control. Plate C showsthat a 0.0078% Daconil®+enzyme disk treatment resulted in a 97%reduction in growth relative to the non-treated control, and a 32%synergistic effect compared to the additive effects of fungicide-onlyand enzyme disk-only treatments. Plate D shows that an enzyme disk-onlytreatment resulted in a 46% reduction in growth relative to thenon-treated control.

FIG. 7. Reduction in P. aphanidermatum growth following treatment byDaconil®, enzyme disk, and Daconil®+enzyme disk. Enzymes concentrationsin enzyme disks were 81 nM. Plate A shows that a Daconil®-only treatment(0.078% Daconil®) resulted in a 34% reduction in growth relative to thenon-treated control. Plate B shows a non-treated control. Plate C showsthat a 0.078% Daconil®+enzyme disk treatment resulted in a 100%reduction in growth relative to the non-treated control. The small whitehalo surrounding the culture plug is due to the application of Daconil®and is not mycelial growth. Plate D shows that an Enzyme disk-onlytreatment resulted in an 84% reduction in growth relative to thenon-treated control.

FIG. 8. Enhanced activity of ampicillin with dry enzyme disk againstXcv. Enzymes concentrations in enzyme disks were 238 nM. Plate A showsthat 100 μg ampicillin alone resulted in a zone of interference (ZOI) of36 mm and no zone of clearance (ZOC). Sparse ampicillin-resistantcolonies can be seen throughout the ZOI. Plate B shows that a 100 μgampicillin plus dry enzyme disk resulted in a ZOC of 32.5 mm and anadditional ZOI that extended 4.5 mm beyond the edge of the ZOC. Plate Cshows that a dry enzyme disk alone resulted in a ZOC of 33 mm with noZOI beyond the ZOC boundary. Plates A-C are visualized using reverseblack and white imaging to enhance growth visualization.

FIG. 9: Percent of nematodes killed after three-day treatmentincubation. Each bar represents the mean of three replicates.

FIGS. 10A and 10B: Effect of treatments on nematodes hatching from cysts(FIG. 10A) and eggs (FIG. 10B). Black bars show live nematode juvenilescounted and white bars show dead nematode juveniles counted. Each barrepresents the mean of three replicates.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for reducingmicrobial contamination or infection in plants, animals, fabrics, andproducts therefrom. This is accomplished, for the first time, by thesynergy of chemical antimicrobial agents with multicomponentcompositions comprising (1) a hydrogen peroxide producing (HPP) enzymeand a free radical producing (FRP) enzyme in self-assembled magneticnanoparticles in one component and (2) substrates for the enzymes inanother component. These magnetically-immobilized enzymes may be insolid or liquid compositions that are stable or inactive. Thus, they maybe stored prior to or after incorporation into products. When thefungicidal activities are required, these multicomponent compositionsare activated by exposure to hydration and/or oxygen. The HPP enzymeacts on substrates to produce hydrogen peroxide and, e.g.D-glucono-δ-lactone. The FRP enzyme acts on the hydrogen peroxide andone or more further substrates to produce free radicals. The hydrogenperoxide and free radicals have antimicrobial properties. In alternativeembodiments, hydrogen peroxide is provided as opposed to a hydrogenperoxide producing enzyme plus its substrates. The antimicrobialactivities are activated by exposure to hydration and/or oxygen. Thedisclosures of Int'l Pub. Nos. WO2012122437 and WO2014055853 as well asInt'l Appl. No. PCT/US16/31419, incorporated by reference herein intheir entirety.

Self-assembled mesoporous nanoclusters comprising entrapped peroxidasesare highly active and robust. The technology is a powerful blend ofbiochemistry, nanotechnology, and bioengineering at three integratedlevels of organization: Level 1 is the self-assembly of peroxidase andoxidase enzymes with magnetic nanoparticles (MNP) for the synthesis ofmagnetic mesoporous nanoclusters. This level uses a mechanism ofmolecular self-entrapment to immobilize and stabilize enzymes. Level 2is the stabilization of the MNPs into other matrices. Level 3 is productconditioning and packaging for Level 1+2 delivery. The assembly ofmagnetic nanoparticles adsorbed to enzyme is herein also referred to asa “bionanocatalyst” (BNC).

MNP immobilization provides highly active and cost-effectiveperoxidases. Peroxidases are very potent enzymes yet notoriouslydifficult to deploy in industrial settings due to strong inhibition inpresence of excess peroxide. NPs increase peroxidation activity andreduce their inhibition which renders them industrially useful.Additionally, the MNPs allow for a broader range of operating conditionssuch as temperature, ionic strength and pH. The size and magnetizationof the MNPs affect the formation and structure of the NPs, all of whichhave a significant impact on the activity of the entrapped enzymes. Byvirtue of their surprising resilience under various reaction conditions,MNPs can be used as improved enzymatic or catalytic agents where othersuch agents are currently used. Furthermore, they can be used in otherapplications where enzymes have not yet been considered or foundapplicable.

The BNC contains mesopores that are interstitial spaces between themagnetic nanoparticles. The enzymes are preferably embedded orimmobilized within at least a portion of mesopores of the BNC. As usedherein, the term “magnetic” encompasses all types of useful magneticcharacteristics, including permanent magnetic, superparamagnetic,paramagnetic, ferromagnetic, and ferrimagnetic behaviors.

The magnetic nanoparticle or BNC has a size in the nanoscale, i.e.,generally no more than 500 nm. As used herein, the term “size” can referto a diameter of the magnetic nanoparticle when the magneticnanoparticle is approximately or substantially spherical. In a casewhere the magnetic nanoparticle is not approximately or substantiallyspherical (e.g., substantially ovoid or irregular), the term “size” canrefer to either the longest the dimension or an average of the threedimensions of the magnetic nanoparticle. The term “size” may also referto an average of sizes over a population of magnetic nanoparticles(i.e., “average size”).

In different embodiments, the magnetic nanoparticle has a size ofprecisely, about, up to, or less than, for example, 500 nm, 400 nm, 300nm, 200 nm, 100 nm, 50 nm, 40 nm, 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, 5nm, 4 nm, 3 nm, 2 nm, or 1 nm, or a size within a range bounded by anytwo of the foregoing exemplary sizes.

In the BNC, the individual magnetic nanoparticles can be considered tobe primary nanoparticles (i.e., primary crystallites) having any of thesizes provided above. The aggregates of nanoparticles in a BNC arelarger in size than the nanoparticles and generally have a size (i.e.,secondary size) of at least about 5 nm. In different embodiments, theaggregates have a size of precisely, about, at least, above, up to, orless than, for example, 5 nm, 8 nm, 10 nm, 12 nm, 15 nm, 20 nm, 25 nm,30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm,150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, or 800 nm, or asize within a range bounded by any two of the foregoing exemplary sizes.

Typically, the primary and/or aggregated magnetic nanoparticles or BNCsthereof have a distribution of sizes, i.e., they are generally dispersedin size, either narrowly or broadly dispersed. In different embodiments,any range of primary or aggregate sizes can constitute a major or minorproportion of the total range of primary or aggregate sizes. Forexample, in some embodiments, a particular range of primary particlesizes (for example, at least about 1, 2, 3, 5, or 10 nm and up to about15, 20, 25, 30, 35, 40, 45, or 50 nm) or a particular range of aggregateparticle sizes (for example, at least about 5, 10, 15, or 20 nm and upto about 50, 100, 150, 200, 250, or 300 nm) constitutes at least orabove about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the totalrange of primary particle sizes. In other embodiments, a particularrange of primary particle sizes (for example, less than about 1, 2, 3,5, or 10 nm, or above about 15, 20, 25, 30, 35, 40, 45, or 50 nm) or aparticular range of aggregate particle sizes (for example, less thanabout 20, 10, or 5 nm, or above about 25, 50, 100, 150, 200, 250, or 300nm) constitutes no more than or less than about 50%, 40%, 30%, 20%, 10%,5%, 2%, 1%, 0.5%, or 0.1% of the total range of primary particle sizes.

The aggregates of magnetic nanoparticles (i.e., “aggregates”) or BNCsthereof can have any degree of porosity, including a substantial lack ofporosity depending upon the quantity of individual primary crystallitesthey are made of. In particular embodiments, the aggregates aremesoporous by containing interstitial mesopores (i.e., mesopores locatedbetween primary magnetic nanoparticles, formed by packing arrangements).The mesopores are generally at least 2 nm and up to 50 nm in size. Indifferent embodiments, the mesopores can have a pore size of preciselyor about, for example, 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 35, 40, 45,or 50 nm, or a pore size within a range bounded by any two of theforegoing exemplary pore sizes. Similar to the case of particle sizes,the mesopores typically have a distribution of sizes, i.e., they aregenerally dispersed in size, either narrowly or broadly dispersed. Indifferent embodiments, any range of mesopore sizes can constitute amajor or minor proportion of the total range of mesopore sizes or of thetotal pore volume. For example, in some embodiments, a particular rangeof mesopore sizes (for example, at least about 2, 3, or 5, and up to 8,10, 15, 20, 25, or 30 nm) constitutes at least or above about 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% of the total range of mesoporesizes or of the total pore volume. In other embodiments, a particularrange of mesopore sizes (for example, less than about 2, 3, 4, or 5 nm,or above about 10, 15, 20, 25, 30, 35, 40, 45, or 50 nm) constitutes nomore than or less than about 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, 0.5%,or 0.1% of the total range of mesopore sizes or of the total porevolume.

The magnetic nanoparticles can have any of the compositions known in theart. In some embodiments, the magnetic nanoparticles are or include azerovalent metallic portion that is magnetic. Some examples of suchzerovalent metals include cobalt, nickel, and iron, and their mixturesand alloys. In other embodiments, the magnetic nanoparticles are orinclude an oxide of a magnetic metal, such as an oxide of cobalt,nickel, or iron, or a mixture thereof. In some embodiments, the magneticnanoparticles possess distinct core and surface portions. For example,the magnetic nanoparticles may have a core portion composed of elementaliron, cobalt, or nickel and a surface portion composed of a passivatinglayer, such as a metal oxide or a noble metal coating, such as a layerof gold, platinum, palladium, or silver. In other embodiments, metaloxide magnetic nanoparticles or aggregates thereof are coated with alayer of a noble metal coating. The noble metal coating may, forexample, reduce the number of charges on the magnetic nanoparticlesurface, which may beneficially increase dispersibility in solution andbetter control the size of the BNCs. The noble metal coating protectsthe magnetic nanoparticles against oxidation, solubilization by leachingor by chelation when chelating organic acids, such as citrate, malonate,or tartrate, are used in the biochemical reactions or processes. Thepassivating layer can have any suitable thickness, and particularly, atleast, up to, or less than, about for example, 0.1 nm, 0.2 nm, 0.3 nm,0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1 nm, 2 nm, 3 nm, 4 nm,5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm, or a thickness in a rangebounded by any two of these values.

Magnetic materials useful for the invention are well-known in the art.Non-limiting examples comprise ferromagnetic and ferromagnetic materialsincluding ores such as iron ore (magnetite or lodestone), cobalt, andnickel. In other embodiments, rare earth magnets are used. Non-limitingexamples include neodymium, gadolinium, dysprosium, samarium-cobalt,neodymium-iron-boron, and the like. In yet further embodiments, themagnets comprise composite materials. Non-limiting examples includeceramic, ferrite, and alnico magnets. In preferred embodiments, themagnetic nanoparticles have an iron oxide composition. The iron oxidecomposition can be any of the magnetic or superparamagnetic iron oxidecompositions known in the art, e.g., magnetite (FesO/O, hematite (α-Fe2θ3), maghemite (γ-Fe2C>3), or a spinel ferrite according to the formulaAB₂O₄, wherein A is a divalent metal (e.g., Xn²⁺, Ni²⁺, Mn²⁺, Co²⁺,Ba²⁺, Sr²⁺, or combination thereof) and B is a trivalent metal (e.g.,Fe³⁺, Cr³⁺, or combination thereof).

The individual magnetic nanoparticles or aggregates thereof or BNCsthereof possess any suitable degree of magnetism. For example, themagnetic nanoparticles, BNCs, or BNC scaffold assemblies can possess asaturated magnetization (Ms) of at least or up to about 5, 10, 15, 20,25, 30, 40, 45, 50, 60, 70, 80, 90, or 100 emu/g. The magneticnanoparticles, BNCs, or BNC-scaffold assemblies preferably possess aremanent magnetization (Mr) of no more than (i.e., up to) or less than 5emu/g, and more preferably, up to or less than 4 emu/g, 3 emu/g, 2emu/g, 1 emu/g, 0.5 emu/g, or 0.1 emu/g. The surface magnetic field ofthe magnetic nanoparticles, BNCs, or BNC-scaffold assemblies can beabout or at least, for example, about 0.5, 1, 5, 10, 50, 100, 200, 300,400, 500, 600, 700, 800, 900, or 1000 Gauss (G), or a magnetic fieldwithin a range bounded by any two of the foregoing values. Ifmicroparticles are included, the microparticles may also possess any ofthe above magnetic strengths.

The magnetic nanoparticles or aggregates thereof can be made to adsorb asuitable amount of enzyme, up to or below a saturation level, dependingon the application, to produce the resulting BNC. In differentembodiments, the magnetic nanoparticles or aggregates thereof may adsorbabout, at least, up to, or less than, for example, 1, 5, 10, 15, 20, 25,or 30 pmol/m2 of enzyme. Alternatively, the magnetic nanoparticles oraggregates thereof may adsorb an amount of enzyme that is about, atleast, up to, or less than, for example, about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% of a saturation level.

The magnetic nanoparticles or aggregates thereof or BNCs thereof possessany suitable pore volume. For example, the magnetic nanoparticles oraggregates thereof can possess a pore volume of about, at least, up to,or less than, for example, about 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3,0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95,or 1 cm3/g, or a pore volume within a range bounded by any two of theforegoing values. [0052] The magnetic nanoparticles or aggregatesthereof or BNCs thereof possess any suitable specific surface area. Forexample, the magnetic nanoparticles or aggregates thereof can have aspecific surface area of about, at least, up to, or less than, forexample, about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, or 200 m 2/g.

MNPs, their structures, organizations, suitable enzymes, and uses aredescribed in WO2012122437 and WO2014055853, incorporated by referenceherein in their entirety.

The compositions and methods of the invention, among other things,reduce or eliminate plant death due to pathogens. In some embodiments,the invention reduces or eliminates “damping off” The AmericanPhytopathological Society defines damping-off as “the death of aseedling before or shortly after emergence due to decomposition of theroot and/or lower stem; it is common to distinguish betweenpre-emergence damping-off and post-emergence damping-off. Pre-emergencedamping-off occurs before a seedling emerges from the soil line.Post-emergence damping-off occurs shortly after a seedling emerges fromthe soil line. The disease is commonly caused by the fungus Rhizoconiasolani and numerous species in the oomycete genus Pythium, althoughother fungi and oomycetes can contribute. The disease is notcrop-specific and causes losses to all agricultural crops.

In other embodiments, the assemblies have antimicrobial propertiesagainst a wide array of pathogens. In some embodiments, the pathogensinclude pathogenic plant bacteria species such as Acidovorax avenae,Agrobacterium tumefaciens, Burkholderia andropogonis, Burkholderiacaryophylli, Burkholderia glumae, Candidatus Liberibacter, CandidatusPhytoplasma solani, Clavibacter michiganensis, Dickeya dadantii, Erwiniapsidii, Pectobacterium atrosepticum, Pectobacterium betavasculorum,Pectobacterium carotovorum, Pectobacterium carotovorum subsp.betavasculorum, Pectobacterium wasabiae, Phytoplasma, Pseudomonasamygdali, Pseudomonas asplenii, Pseudomonas caricapapayae, Pseudomonascichorii, Pseudomonas coronafaciens, Pseudomonas corrugate, Pseudomonasficuserectae, Pseudomonas flavescens, Pseudomonas fuscovaginae,Pseudomonas helianthi, Pseudomonas marginalis, Pseudomonasoryzihabitans, Pseudomonas palleroniana, Pseudomonas papaveris,Pseudomonas salomonii, Pseudomonas savastanoi, Pseudomonas syringae,Pseudomonas tomato, Pseudomonas turbinellae, Pseudomonas viridiflava,Psyllid yellows, Ralstonia solanacearum, Rhodococcus fascians,Spiroplasma citri, Xanthomonas axonopodis, Xanthomonas campestris,Xanthomonas oryzae, Xylella fastidiosa, Escherichia coli, Salmonellaenterica, Listeria monocytogenes, and other plant, animal, human,soilborne, and environmental pathogens.

In other embodiments, the assemblies have antimicrobial propertiesagainst non-plant pathogen bacteria including Escherishia Coli, Brucellasp., Vibrio sp., Serratia sp., Nocardia sp., Leptospira sp.,Mycobacterium sp., Clostridium sp., Bacillus sp., Pseudomonas sp.Staphylococcus sp., Neisseria sp., Haemophilus sp., Helicobacter sp.,Mycoplasma sp., Pseudomonas sp. Treponema sp., and Yersinia sp.

In other embodiments, the fungicidal assemblies are effective againstplant pathogenic fungi including genera such as Alternaria sp.,Armillaria sp. Ascochyta sp., Aspergillus sp., Bipoloaris, Bjerkanderasp., Botrytis sp., Ceratobasidium sp., Cercospora sp., Chrysimyxa sp.,Cladosporium sp., Cochliobolus sp., Coleosporium sp., Colletotrichumsp., Cylindrocladium sp., Cytospora sp., Diaporthe sp., Didymella sp.,Drechslera sp., Erysiphe sp, Exobasidium sp., Fusarium sp., Ganodermasp., Gibberella sp., Gymnospragium sp., Helicobasidium sp., Inonotussp., Leptosphaeria sp., Leucostoma sp. Marasmius sp., Microspaera sp.,Mucor sp., Mycosphaerella sp., Nectria sp., Oidium sp., Passalora sp.,Pestalotiopsis sp., Phaeoramularia sp., Phoma sp., Phyllosticta sp.,Pseudocercospora sp., Puccinia sp., Pyrenophora sp., Rhizoctonia sp.,Rhizopus sp., Septoria sp., Sphaceloma sp., Stemphylium sp., Stigminasp., Tilletia sp., Typhula sp., Uromyces sp., Ustilago sp., andVerticillium sp.

In other embodiments, the fungicidal assemblies are effective againstplant pathogenic oomycetes including genera such as Aphanomyces sp.,Bremia sp., Peronosclerospora sp., Peronospora sp., Phytophthora sp.,Plasmopara sp., Pseudoperonospora sp., Pythium sp. and Sclerophthora sp.In preferred embodiments, the oomycetes are Phytophthora infestans,Hyaloperonospora arabidopsidis, Phytophthora ramorum, Phytophthorasojae, Phytophthora capsici, Plasmopara viticola, Phytophthoracinnamomi, Phytophthora parasitica, Pythium ultimum, or Albugo candida.

A number of genera and species of nematodes are highly damaging to agreat range of plants, including foliage plants, agronomic and vegetablecrops, fruit and nut trees, turfgrass, and forest trees. Thus, in someembodiments, the assemblies of the invention are effective againstnematodes such as Meloidogyne species (spp.), Heterodera spp., Globoderaspp., Pratylenchus spp., Helicotylenchus spp., Radopholus similis,Ditylenchus dipsaci, Rotylenchulus reniformis, Xiphinema spp,Aphelenchoides spp., Toxocara spp., Bursaphelenchus xylophilus, andtrichinella spiralis.

In other embodiments, the invention is effective against plant virusesthat include plant viruses such as Mosaic Viruses, Mottle Viruses,Begomoviruses, Carlaviruses, Carmoviruses, Criniviruses, Fabaviruses,Furoviruses, Machlomoviruses, Macluraviruses, Necroviruses,Potexviruses, Tenuiviruses, and Tospoviruses.

In some embodiments, the invention provides hydrogen peroxide producing(HPP) enzymes. In certain embodiments, the HPP enzymes are oxidases thatmay be of the EX 1.1.3 subgenus. In particular embodiments, the oxidasemay be EC 1.1.3.3 (malate oxidase), EC 1.1.3.4 (glucose oxidase), EC1.1.3.5 (hexose oxidase), EC 1.1.3.6 (cholesterol oxidase), EC 1.1.3.7(aryl-alcohol oxidase), EC 1.1.3.8 (L-gulonolactone oxidase), EC 1.1.3.9(galactose oxidase), EC 1.1.3.10 (pyranose oxidase), EC 1.1.3.11(L-sorbose oxidase), EC 1.1.3.12 (pyridoxine 4-oxidase), EC 1.1.3.13(alcohol oxidase), EC 1.1.3.14 (catechol oxidase), EC 1.1.3.15(2-hydroxy acid oxidase), EC 1.1.3.16 (ecdysone oxidase), EC 1.1.3.17(choline oxidase), EC 1.1.3.18 (secondary-alcohol oxidase), EC 1.1.3.19(4-hydroxymandelate oxidase), EC 1.1.3.20 (long-chain alcohol oxidase),EC 1.1.3.21 (glycerol-3-phosphate oxidase), EC 1.1.3.22, EC 1.1.3.23(thiamine oxidase), EC 1.1.3.24 (L-galactonolactone oxidase), EC1.1.3.25, EC 1.1.3.26, EC 1.1.3.27 (hydroxyphytanate oxidase), EC1.1.3.28 (nucleoside oxidase), EC 1.1.3.29 (Nacylhexosamine oxidase), EC1.1.3.30 (polyvinyl alcohol oxidase), EC 1.1.3.31, EC 1.1.3.32, EC1.1.3.33, EC 1.1.3.34, EC 1.1.3.35, EC 1.1.3.36, EC 1.1.3.37D-arabinono-1,4-lactone oxidase), EC 1.1.3.38 (vanillyl alcoholoxidase), EC 1.1.3.39 (nucleoside oxidase, H₂O₂ forming), EC 1.1.3.40(D-mannitol oxidase), or EC 1.1.3.41 (xylitol oxidase).

The invention provides Free Radical Producing (FRP) enzymes in one ofthe sequential components of the solid fungicidal compositions. In someembodiments, the FRP is a peroxidase. Peroxidases are widely found inbiological systems and form a subset of oxidoreductases that reducehydrogen peroxide (H₂O₂) to water in order to oxidize a large variety ofaromatic compounds ranging from phenol to aromatic amines.

Peroxidases belong to the sub-genus EC 1.11.1. In certain embodiments,the EC 1.11.1 enzyme is The EC 1.11.1 enzyme can be more specifically,for example, EC 1.11.1.1 (NADH peroxidase), EC 1.11.1.2 (NADPHperoxidase), EC 1.11.1.3 (fatty acid peroxidase), EC 1.11.1.4, EC1.11.1.5 (cytochrome-c peroxidase), EC 1.11.1.6 (catalase), EC 1.11.1.7(peroxidase), EC 1.11.1.8 (iodide peroxidase), EC 1.11.1.9 (glutathioneperoxidase), EC 1.11.1.10 (chloride peroxidase), EC 1.11.1.11(L-ascorbate peroxidase), EC 1.11.1.12 (phospholipid-hydroperoxideglutathione peroxidase), EC 1.11.1.13 (manganese peroxidase), EC1.11.1.14 (diarylpropane peroxidase), or EC 1.11.1.15 (peroxiredoxin).

In other embodiments, the peroxidase may also be further specified byfunction, e.g., a lignin peroxidase, manganese peroxidase, or versatileperoxidase. The peroxidase may also be specified as a fungal, microbial,animal, or plant peroxidase. The peroxidase may also be specified as aclass I, class II, or class III peroxidase. The peroxidase may also bespecified as a myeloperoxidase (MPO), eosinophil peroxidase (EPO),lactoperoxidase (LPO), thyroid peroxidase (TPO), prostaglandin Hsynthase (PGHS), glutathione peroxidase, haloperoxidase, catalase,cytochrome c peroxidase, horseradish peroxidase, peanut peroxidase,soybean peroxidase, turnip peroxidase, tobacco peroxidase, tomatoperoxidase, barley peroxidase, or peroxidasin. In these particularembodiments, the peroxidase is a lactoperoxidase.

The lactoperoxidase/glucose oxidase (LP/GOX) antimicrobial system occursnaturally in bodily fluids such as milk, saliva, tears, and mucous(Bosch et al., J. Applied Microbiol., 89(2), 215-24 (2000)). This systemutilizes thiocyanate (SCN—) and iodide (I—), two naturally occurringcompounds that are harmless to mammals and higher organisms (Welk et al.Archives of Oral Biology, 2587 (2011)). LP catalyzes the oxidation ofthiocyanate and iodide ions into hypothiocyanite (OSCN—) and hypoiodite(OI—), respectively, in the presence of hydrogen peroxide (H₂O₂). TheH₂O₂ in this system is provided by the activity of GOX on β-D-glucose inthe presence of oxygen. These free radical compounds, in turn, oxidizesulfhydryl groups in the cell membranes of microbes (Purdy, Tenovuo etal. Infection and Immunity, 39(3), 1187 (1983); Bosch et al., J. AppliedMicrobiol., 89(2), 215-24 (2000), leading to impairment of membranepermeability (Wan, Wang et al. Biochemistry Journal, 362, 355-362(2001)) and ultimately microbial cell death. Concentrations as low as 20μM of hypothiocyanite and hypoiodite can result in inhibition of cellgrowth (Bosch, van Doorne et al. 2000). The LP/GOX system is effectiveon thiocyanate on its own; when paired with iodide, there is asynergistic effect that enhances biostatic and biocidal activity andextends the susceptible target range including Gram negative bacteria(e.g., E. coli, P. aerugenosa), Gram positive bacteria (e.g., S. aureus,Streptococcus spp.), and fungus (e.g., C. albicans) (Reiter, Marshall etal. Infection and Immunity, 13(3), 800-807 (1976); Bosch et al., J.Applied Microbiol., 89(2), 215-24 (2000); Welk et al. Archives of OralBiology, 2587 (2011).) Furthermore, the LP/GOX system functions in twophases: (1) the generation and action of hypothiocyanite and hypoioditeon cell membranes, and then, when these compounds are depleted, (2)excess H₂O₂ builds up, enacting its own oxidative damage on cellularstructures (Reiter, Marshall et al. 1976). The forgoing references areincorporated herein by reference in their entirety.

The enzyme system has been deployed and approved in the industry forbiofilm control such as toothpaste and milk anti-spoiling agents. Thesystem is largely non-specific and robust with few reactionrequirements. One study found persistent biostatic and biocidal activityagainst Gram (−) and (+) bacteria and C. albicans after 18 months ofre-inoculation every two months Bosch et al., J. Applied Microbial.,89(2), 215-24 (2000). The effective pH range is 3-7 with a peak LPactivity at pH 5 (Reiter, Marshall et al. 1976; Purdy, Tenovuo et al.1983). Higher activity is typically witnessed against bacteria at pH 3,but this is likely due to inhibition of growth by low pH (Reiter,Marshall et al. 1976). Other than pH, the only strict requirement foractivity of the LP/GOX system is the presence of oxygen, without whichGOX can't generate H₂O₂ from glucose. The forgoing references areincorporated herein by reference in their entirety.

LP/GOX has been described as a pesticide for microorganisms that includebacteria and fungi. (See U.S. Pat. No. 6,447,811, incorporated byreference herein in its entirety). Thus, in some embodiments, theinvention described herein provides magnetically-immobilized pesticidesin solid or liquid formulations. The pesticides comprise a peroxidaseenzyme that produces a free radical. In some embodiments, the peroxidaseenzyme is lactoperoxidase. The pesticides further comprise a peroxidesource that may include an enzyme that oxidizes glucose.

In some embodiments of the invention, the chemical fungicide may be oneor more of the following: mefenoxam, myclobutanil, chlorothalonil,prothioconazole, trifloxystrobin, propiconazole, mancozeb, Copper,methyl benzimidazole carbamates, dicarboximides, demethylationinhibitors (DMI), phenylamides (PA), amines, phosphorothiolates,dithiolanes, carboxamides, hydroxy-(2-amino-)pyrimidines,anilino-pyrimidines (AP), N-phenyl carbamates, quinone outsideinhibitors (QOI), phenylpyrroles (PP), quinolines, aromatic hydrocarbons(AH), heteroaromatics, melanin biosynthesis inhibitors-dehydratase(MBI-D), hydroxyanilides, succinate biosynthesis inhibitors (SBI),polyoxins, phenylureas, quinone inside inhibitors (QiI), benzamides,enopyranuronic acid antibiotic, hexopyranosyl antibiotic, glucopyranosylantibiotic, cyanoacetamide-oximes, carbamates, uncouplers of oxidativephosphorylation, organo tin compounds, carboxylic acids,heteroaromatics, phosphonates, phthalamic acids, benzotriazines,benzene-sulfonomides, pyridazinones, ATP production inhibitors, complexI of respiration inhibitors, carboxylix acid amides (CAA), tetracyclineantibiotic, thiocarbamate, host plant defense inducers includingsalicylic acid pathway, fungicides with unknown target sites of action,fungicides with multi-site contact activity, mineral oils, organic oils,or potassium bicarbonate.

In some embodiments of the invention, a chemical antibiotic is used thatmay be one or more of the following: chemical families ofaminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins,glycopeptides, lincosamides, lipopeptides, macrolides, monolactams,nitrofurans, oxazolidinones, penicillins, polypeptide antibiotics,quinolones, fluoroquinolones, sulfonamides, tetracyclines.aminoglycosides, ansamycins, carbapenems, cephalosporins, glycopeptides,lincosamides, lipopeptides, macrolides, monobactams, nitrofurans,oxazolidinones, penicillins, polypeptides, quinolones, rifamycins,streptogramins, sulfonamides, tetracyclines, tuberactinomycins, or drugswith activity against mycobacteria. In preferred embodiments, thechemical antibiotic is ampicillin.

The invention provides that the chemical fungicides and antibiotics(chemical microbiocides) may be measured by its minimum inhibitoryconcentration (MIC) in the compositions and methods described herein.The MIC is the lowest concentration of a chemical that prevents visiblegrowth of a bacterium, fungus, or oomycete. The MIC of the microbiocidesmay be determined, for instance, by preparing solutions of the chemicalat increasing concentrations, incubating the solutions with the separatebatches of cultured bacteria, and measuring the results using agardilution or broth microdilution

The minimum bactericidal concentration (MBC) is the lowest concentrationof an antibacterial agent required to kill a particular bacterium. Itcan be determined from broth dilution minimum inhibitory concentration(MIC) tests by subculturing on agar plates that do not contain the testagent. The MBC is identified by determining the lowest concentration ofantibacterial agent that reduces the viability of the initial bacterialinoculum by ≥99.9%. The MBC is complementary to the MIC; whereas the MICtest demonstrates the lowest level of antimicrobial agent that inhibitsgrowth, the MBC demonstrates the lowest level of antimicrobial agentthat results in microbial death. This means that even if a particularMIC shows inhibition, plating the bacteria onto agar might still resultin organism proliferation because the antimicrobial did not cause death.Antibacterial agents are usually regarded as bactericidal if the MBC isno more than four times the MIC. Microorganisms may survivemicrobiocides because they develop resistance to them.

The final chemical fungicide or antibiotic in the invention is at afinal concentration of less than 1% or about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9% 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the minimuminhibitory concentration (MIC) or minimum bactericidal concentration(MBC).

The chemical microbiocides in the invention can also be measured bytheir EC₅₀. The term half maximal effective concentration (EC₅₀) refersto the concentration of a drug, antibody or toxicant which induces aresponse halfway between the baseline and maximum after a specifiedexposure time. It is used herein as a measure of microbiocide potency.The EC₅₀ of a graded dose response curve therefore represents theconcentration of a compound where 50% of its maximal effect is observed.The EC₅₀ of a quantal dose response curve represents the concentrationof a compound where 50% of the population exhibit a response after aspecified exposure duration. The microbiocides in the invention,including the fungicides and antibiotics, is at a final concentration ofless than 1% or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%. In other embodiments, the finalchemical microbiocidal concentration is between about 100% and 500%,500% and 1000%, 1000% and 2000%, 2000% and 2500%, 2500% and 5000%, 5000%and 10,000%.

The invention provides inactive magnetically-immobilized enzymes. Theenzymes may be inactive because they are not exposed to water, oxygen,substrates, or any combination thereof. In a preferred embodiment of thepresent invention, the magnetically-immobilized enzymes are in an oilbase. This limits enzymatic activity prior to use. Activation of theimmobilized enzymes occurs upon exposure to hydration and/or oxygen. Ina more preferred embodiment, the magnetically-immobilized enzymes are inan oil base comprising an agent for emulsifying the oil in an aqueoussolution to form an oil-in-water emulsion. In another more preferredembodiment, the oil is a mineral oil, vegetable oil, or animal oil.Exemplary mineral oils include paraffin oil and kerosene-type oils.Exemplary animal oils include fish oils such as herring and mackereloil. Examples of vegetable oils are peanut oil, sesame oil, rape-seedoil, linseed oil, castor oil, soybean oil, corn germ oil, andcotton-seed oil.

In other embodiments, in order to further facilitate the distribution ofthe magnetically-immobilized enzymes over a surface, one or morespreading agents known in the art can further be added to thecomposition or the oil base. In some embodiments, the spreading agentsare non-ionogenic surface tension-reducing substances. In preferredembodiments, the spreading agents are ethoxylated alcohols andphosphatidyl lipids.

In other embodiments, one or more adhesives can be added. Adhesives mayhelp prevent the magnetically-immobilized enzymes from being rinsed offthe plant by rain or other conditions. Adhesives are well known in theart. Examples are starch, gums such as xanthan gum, gum Arabic andcarboxymethyl celluloses (CMCs).

The composition can be applied by means of coating, spraying,sprinkling, atomizing, overhead spraying, watering, immersing, and dripirrigation. A particularly advantageous method for applying thecomposition is spraying both by means of low volume methods (mistsystems) and high volume methods. Drip irrigation can be used forculture systems on rockwool and other growth substrates. Themagnetically-immobilized enzymes according to the invention can also beused to disinfect drip irrigation systems. In both latter cases thepresence of the oil base is not strictly necessary for an optimalactivity. Immersion in a bath with the composition is particularlysuitable for the treatment of plant parts, in particular harvestableparts, such as bulbs, tubers, fruits and the like.

The magnetically-immobilized enzymes can be made commercially availablein different forms. In a preferred embodiment, the peroxidase activityis delayed as long as possible because this increases the shelf-life ofthe product. The enzymatic activity starts upon exposure to bothhydration (i.e. water) and oxygen. In the present case the glucoseoxidase/glucose system is the hydrogen peroxide donor. In more preferredembodiments, the hydrogen peroxide donor is provided separately from theperoxidase. In addition, the oil base and the spreading agent can, ifdesired, also be packaged separately.

In another embodiment, a kit is provided for forming the composition thekit comprises an optionally concentrated enzyme composition comprising aperoxidase (e.g. lactoperoxidase) and a hydrogen peroxide donor (e.g.glucose oxidase and glucose). In preferred embodiments, the kit mayfurther comprise thiocyanate, iodide, oil, an emulsifier, or spreadingagents. In more preferred embodiments, the ingredients are mixed witheach other before use. In another embodiment, the kit may comprise oneor more ingredients in a concentrated form for dilution or hydrationprior to or concurrently with use.

In embodiments where β-D-Glucose is oxidized to H₂O₂, or where cellulosederived sugars are oxidized to H₂O₂, cellulase enzymes may be providedwith the compositions of the invention. In some embodiments, the seedcoating further comprises the cellulase.

In some embodiments, the cellulases are exocellulases, endocellulases,hemicellulases, or combinations thereof known in the art. Endocellulase(EC 3.2.1.4) randomly cleaves internal bonds at amorphous sites thatcreate new chain ends. Exocellulase (EC 3.2.1.91) cleaves two to fourunits from the ends of the exposed chains produced by endocellulase,resulting in the tetrasaccharides or disaccharides, such as cellobiose.There are two main types of exocellulases [or cellobiohydrolases (CBH)]—CBHI works processively from the reducing end, and CBHII worksprocessively from the nonreducing end of cellulose. Cellobiase (EC3.2.1.21) or beta-glucosidase hydrolyses the exocellulase product intoindividual monosaccharides. Oxidative cellulases depolymerize celluloseby radical reactions, as for instance cellobiose dehydrogenase(acceptor). Cellulose phosphorylases depolymerize cellulose usingphosphates instead of water.

In other embodiments, endocellulases may include EC 3.2.1.4,endo-1,4-beta-D-glucanase, beta-1,4-glucanase, beta-1,4-endoglucanhydrolase, celluase A, cellulosin AP, endoglucanase D, alkali cellulase,cellulase A 3, celludextrinase, 9.5 cellulase, avicelase, pancellase SS,and 1,4-(1,3, 1,4)-beta-D-glucan 4-glucanohydrolase). Cellulases enzymesare typically produced by fungi, bacteria, and protozoans of cellulose).Other names for ‘endoglucanases’ are: endo-1,4-beta-glucanase,carboxymethyl cellulase (CMCase), endo-1,4-beta-D-glucanase,beta-1,4-glucanase, beta-1,4-endoglucan hydrolase, and celludextrinase.

In some embodiments, the methods described herein use recombinant cellsthat express the enzymes used in the invention. Recombinant DNAtechnology is known in the art. In some embodiments, cells aretransformed with expression vectors such as plasmids that express theenzymes. In other embodiments, the vectors have one or more geneticsignals, e.g., for transcriptional initiation, transcriptionaltermination, translational initiation and translational termination.Here, nucleic acids encoding the enzymes may be cloned in a vector sothat it is expressed when properly transformed into a suitable hostorganism. Suitable host cells may be derived from bacteria, fungi,plants, or animals as is well-known in the art.

In some embodiments, the invention provides that the matrix material isa biopolymer. Examples include the polysaccharides (e.g., cellulose,hemicellulose, xylan, chitosan, inulin, dextran, agarose, and alginicacid), polylactic acid, and polyglycolic acid. In other embodiments, thematrix material is a water-soluble cellulose derivative, awater-solvatable cellulose derivative, an alginate derivative, and achitosan derivative.

In some embodiments, the matrix comprises cellulose. Cellulose is anorganic compound with the formula (C₆H₁₀O₅)n, a polysaccharideconsisting of a linear chain of several hundred to many thousands ofβ(1→4) linked D-glucose units. The cellulose used in the invention maybe obtained or derived from plant, algal, or microbial sources. In someembodiments, the invention provides cellulose derivatives known in theart. The hydroxyl groups (—OH) of cellulose can be partially or fullyreacted with reagents known in the art. In preferred embodiments, thecellulose derivatives are cellulose esters and cellulose ethers (—OR).In more preferred embodiments, the cellulose derivatives are celluloseacetate, cellulose triacetate, cellulose proprionate, cellulose acetateproprionate (CAP), cellulose acetate butyrate (CAB), nitrocellulose(cellulose nitrate), cellulose sulfate, methylcellulose, ethylcellulose,ethyl methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose(HPC), hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose(HPMC), ethyl hydroxyethyl cellulose, and carboxymethyl cellulose (CMC).

In some embodiments, the matrix comprises carboxymethyl cellulose.Carboxymethyl cellulose (CMC) or cellulose gum[1] is a cellulosederivative with carboxymethyl groups (—CH2-COOH) bound to some of thehydroxyl groups of the glucopyranose monomers that make up the cellulosebackbone. It is synthesized using techniques known in the art, e.g., bythe alkali-catalyzed reaction of cellulose with chloroacetic acid. Thepolar (organic acid) carboxyl groups render the cellulose soluble andchemically reactive. The functional properties of CMC depend on thedegree of substitution of the cellulose structure (i.e., how many of thehydroxyl groups have taken part in the substitution reaction), as wellas the chain length of the cellulose backbone structure and the degreeof clustering of the carboxymethyl substituents.

In some embodiments, the matrix comprises hydroxypropyl cellulose (HPC).HPC is a derivative of cellulose with both water solubility and organicsolubility. HPC is an ether of cellulose in which some of the hydroxylgroups in the repeating glucose units have been hydroxypropylatedforming —OCH2CH(OH)CH3 groups using propylene oxide. The average numberof substituted hydroxyl groups per glucose unit is referred to as thedegree of substitution (DS). Complete substitution would provide a DS of3. Because the hydroxypropyl group added contains a hydroxyl group, thiscan also be etherified during preparation of HPC. When this occurs, thenumber of moles of hydroxypropyl groups per glucose ring, moles ofsubstitution (MS), can be higher than 3. Because cellulose is verycrystalline, HPC must have an MS about 4 in order to reach a goodsolubility in water. HPC has a combination of hydrophobic andhydrophilic groups, so it has a lower critical solution temperature(LCST) at 45° C. At temperatures below the LCST, HPC is readily solublein water; above the LCST, HPC is not soluble. HPC forms liquid crystalsand many mesophases according to its concentration in water. Suchmesophases include isotropic, anisotropic, nematic and cholesteric. Thelast one gives many colors such as violet, green and red.

In some embodiments, the matrix comprises methyl cellulose. Methylcellulose (or methylcellulose) is derived from cellulose. It is ahydrophilic white powder in pure form and dissolves in cold (but not inhot) water, forming a clear viscous solution or gel. Methyl cellulosedoes not occur naturally and is synthetically produced by heatingcellulose with caustic solution (e.g. a solution of sodium hydroxide)and treating it with methyl chloride. In the substitution reaction thatfollows, the hydroxyl residues (—OH functional groups) are replaced bymethoxide (—OCH₃ groups).

Different kinds of methyl cellulose can be prepared depending on thenumber of hydroxyl groups substituted. Cellulose is a polymer consistingof numerous linked glucose molecules, each of which exposes threehydroxyl groups. The Degree of Substitution (DS) of a given form ofmethyl cellulose is defined as the average number of substitutedhydroxyl groups per glucose. The theoretical maximum is thus a DS of3.0, however more typical values are 1.3-2.6.

In some embodiments, the matrix comprises alginate. Alginate, alsocalled Alginic acid, and algin, is an anionic polysaccharide distributedwidely in the cell walls of brown algae. When bound with water it formsa viscous gum. In extracted form it absorbs water quickly; it is capableof absorbing 200-300 times its own weight in water. It is sold infilamentous, granular or powdered forms. The invention provides matrixmaterials of known alginate and alginate-derived materials. In preferredembodiments, the alginate-derived materials includealginate-polylysine-alginate (APA),Alginate/Poly-l-lysine/Pectin/Poly-l-lysine/Alginate (APPPA),Alginate/Poly-l-lysine/Pectin/Poly-l-lysine/Pectin (APPPP), andAlginate/Poly-L-lysine/Chitosan/Poly-l-lysine/Alginate (APCPA),alginate-polymethylene-co-guanidine-alginate (A-PMCG-A),hydroxymethylacrylate-methyl methacrylate (HEMA-MMA), multilayeredHEMA-MMA-MAA, polyacrylonitrile-vinylchloride (PAN-PVC).

In some embodiments, the matrix comprises chitosan. Chitosan is a linearpolysaccharide composed of randomly distributed β-(1-4)-linkedD-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylatedunit). The amino group in chitosan has a pKa value of ˜6.5, which leadsto a protonation in acidic to neutral solution with a charge densitydependent on pH and the % DA-value. This makes chitosan water solubleand a bioadhesive which readily binds to negatively charged surfacessuch as mucosal membranes. It is produced commercially by deacetylatingchitin, which is the structural element in the exoskeleton ofcrustaceans (such as crabs and shrimp) and cell walls of fungi, withsodium hydroxide. Chitosan is used in agriculture as a seed treatmentand biopesticide. In winemaking, it is used as a fining agent, alsohelping to prevent spoilage. It is also used in bandages to reducebleeding and as an antibacterial agent. It is also be used to helpdeliver drugs through the skin.

In other embodiments, the matrix materials may be acrylonitrile/sodiummethallylsuflonate, (AN-69), polyethylene glycol/polypentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), polyJVjiV-dimethyl acrylamide (PDMAAm), siliceous encapsulates, andcellulose sulphate/sodium alginate/polymethylene-co-guanidine(CS/A/PMCG).

In some embodiments, the invention provides antimicrobial compositionsthat are used, inter alia, for seed coatings. Any seeds that arevulnerable to pathogens that respond to the enzyme systems disclosedherein would benefit. In some embodiments, the seeds may be forvegetables, fruits, field crops, and flowers. In other embodiments, theinvention provides antimicrobial compositions that are used, inter alia,for bedding for industrially or commercially relevant domesticatedanimals and products derived therefrom. Many domesticated animals areknown in the art. In other embodiments, the invention providesfungicidal compositions that are used, inter alia, for wound dressings.Many wound dressings are known in the art. The invention providesfabrics that resist pathogens or contaminants that respond to the enzymesystems disclosed herein. The fabrics comprise the fungicidalcompositions described herein.

Some embodiments of the invention provides compositions and methods forreducing human infections. This is accomplished, for the first time, bya multicomponent composition comprising a hydrogen peroxide producing(HPP) enzyme and a free radical producing (FRP) enzyme in magneticnanoparticles in one component and substrates for the enzymes in anothercomponent. The solid compositions are stable and inactive. Thus, theymay be stored prior to or after incorporation into products. When thefungicidal activities are required, the multicomponent compositions areactivated by hydration. The HPP enzyme acts on substrates to producehydrogen peroxide and D-glucono-δ-lactone. The FRP enzyme acts on thehydrogen peroxide and one or more further substrates to produce freeradicals. The hydrogen peroxide and free radicals have fungicidalproperties.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Example 1—Microbiocidal Optimization of Dry Enzyme and Substrate Disks

Materials and Methods

Five different lactoperoxidase (LP) to glucose oxidase (GOx) molarratios were analyzed in dry enzyme disks to determine the optimal enzymeratio for maximizing bactericidal activity. Two different concentrationsof substrates in the dry substrate disks were also analyzed. Theseanalyses were performed in-vitro on the bacterium Xanthomonas campestrispv. vitians isolate ‘09131A’ which was originally collected in New YorkState and isolated and identified by Christine Smart, Professor of PlantPathology at Cornell University. (http://blogs.cornell.edu/smartlab/.)

A second analysis was done to determine the optimal enzyme concentrationto inhibit fungal growth. Four concentrations of enzymes were analyzedin dry enzyme disks to determine the optimal enzyme concentration tomaximize fungicidal activity. All analyses were performed in-vitro onplant-pathogenic microorganism cultures obtained from collaborators inthe Cornell University Section of Plant Pathology and Plant-MicrobeBiology (Cornell University, Ithaca, N.Y.). The fungi Rhizoctonia solani(isolate ‘AC1-A1’) and Fusarium graminearum (isolate ‘GZ014NY98’) werecollected in New York State isolated and identified by Professors EricNelson and Gary Bergstrom, respectively. The oomycete Pythium ultimum(isolate ‘Geneva16’) was collected in New York State and isolated andidentified by Professor Eric Nelson.https://pppmb.cals.cornell.edu/people/eric-nelson.

A third analysis was done to determine the optimal enzyme concentrationto inhibit oomycete growth. Three concentrations of enzymes wereanalyzed to determine optimal activity against oomycetes. The oomycetePythium ultimum (isolate ‘Geneva16’) was collected in New York State andisolated and identified by Professor Eric Nelson (Cornell University,Ithaca, N.Y.).

For the optimization of enzyme ratios for bactericidal activity,lactoperoxidase (LP) and glucose oxidase (GOx) enzymes were immobilizedin five different LP:GOx molar ratios (see Table 1). Dry enzyme diskcompositions were the same as those listed in Example 2 (Table 2) withthe exception of enzyme concentrations, which are listed in Table 1.Having a 1:1 LP:GOx enzyme disk was considered as a standard, thereforeGOx concentrations were decreased proportionally to create 10:1 and 5:1disks and LP concentrations were decreased proportionally to create the1:5 and 1:10 disks. Magnetic nanoparticles (pH 3) were mixed with LP+GOxenzyme mixtures (pH 7.4) in a 1:1 volume ratio to immobilize enzymes.Nanoparticle concentrations were adjusted to maintain a 30%enzyme:nanoparticle mass ratio (Table 1). Two dry substrate diskformulations that were analyzed, called 1× and 10×, are listed in Table2. Enzyme disk and substrate disk components were mixed with distilleddeionized H₂O to achieve desired concentrations, and dried under vacuumwith a desiccant to remove H₂O. This process was repeated for thecreation of all dry enzyme and substrate disks.

Magnetic nanoparticles were made and used for the molecular entrapmentof peroxidase as described in US20150252352; PCT/US16/31419; and Corgiéet al., Adv. Functional Materials 22:1940-51 (2012). The foregoing areincorporated by reference herein in their entirety.

Analysis to optimize bactericidal efficacy of dry enzyme and substratedisk formulations was performed twice. Each treatment was performed induplicate. There were a total of ten treatments included in the LP: GOxratio optimization. Each of the five LP:GOx ratios was analyzed with 1×and 10× substrate disks. Each analysis also included controls of 1× and10× substrate disks only. The analyses were set up by creating bacteriallawns of Xcv on 85-mm petri dishes containing LB agar. 100 μl of 1×10⁷CFU/ml bacterial suspension was dispensed onto each dish (1×10⁶ CFU perplate), which contained three sterile glass beads. The beads wereswirled to spread the inoculum evenly across the surface of the plate.Dry substrate disks were then placed in the center of each plate usingsterile forceps. Dry enzyme disks were then placed on top of the drysubstrate disks and each plate was wrapped with parafilm and incubatedat 26.7° C. Zones of inhibition surrounding each treatment disk weremeasured after three days. Data were analyzed by analysis of variance inR Studio version 3.3.0 (R Studio, Boston, Mass.) using packages lme4 andlsmeans, and mean separations were done using Tukey's honest significantdifference analysis. Means were considered significantly different atP<0.05.

TABLE 1 Optimization of lactoperoxidase and glucose oxidase in dryenzyme disks LP:GOx LP conc GOx conc. Nanoparticle Enzyme conc. in molarratio (μg/ml) (μg/ml) conc. (mg/ml) dry disks (nM) 10:1  125 25.8 0.50394 5:1 125 51.6 0.589 110 1:1 125 258 1.277 238 1:5 25 258 0.943 176 1:10 12.5 258 0.902 168

TABLE 2 Dry substrate disk compositions Component 1X concentration 10Xconcentration Potassium iodide 0.3 mM 3 mM Ammonium thiocyanate 0.5 mM 5mM Carboxymethyl cellulose 0.7% 0.7% Glucose  50 mM 500 mM 

To optimize enzyme concentrations for activity against fungi, fourenzyme concentrations (1×, 2×, 5×, and 10×) were analyzed in a 1:1 molarratio of LP:GOx. LP at 1250 μg/ml and GOx at 2580 μg/ml were mixed in a1:1 ratio (pH 7.4) and the LP+GOx suspension was combined withnanoparticles at 12.77 mg/ml (pH 3) in a 1:1 enzyme:NP volume ratio.Proportional volumes of immobilized enzymes were then added to enzymedisk components listed in Example 2 (see Table 3) to achieve thefollowing enzyme concentrations: 1×=238 nM; 2×=476 nM; 5×=1190 nM; and10×=2380 nM. Disks were dried under vacuum prior to use.

The dry enzyme disks were analyzed for efficacy against R. solani and F.graminearum. 10× substrate disks (Table 2) were placed on the center of85-mm plates containing potato dextrose agar. Dry enzyme disks were thenplaced on top of substrate disks, one per plate, followed by a 7 mmfungal culture plug placed directly on top of the enzyme disk myceliaside down. Fungal colony diameters were measured after five days (R.solani) and six days (F. graminearum).

To optimize activity against oomycetes, three enzyme concentrations(0.0017×, 0.34×, 1× in a 1:1 molar ratio of LP:GOx) were analyzed.Enzymes were immobilized as previously described and proportionalvolumes of immobilized enzymes were then added to enzyme disk componentslisted in Example 2 (Table 3) to achieve the following enzymeconcentrations: 0.0034×=0.8 nM; 0.68×=161 nM; and 2×=476 nM. Disks weredried prior to use.

The dry enzyme disks were analyzed for efficacy against P. ultimum. 10×substrate disks (see Table 2) were placed on the center of 85-mm platescontaining potato dextrose agar. Dry enzyme disks were then placed ontop of substrate disks, one per plat, followed by a 7 mm fungal cultureplug placed directly on top of the enzyme disk mycelia side down.Oomycete colony diameters were measured after five days.

Bactericidal Enzyme Ratio Optimization

When LP:GOx ratios were analyzed using the 1× substrate concentration,the 1:1 ratio and ratios favoring higher GOx concentrations resulted inthe largest zones of inhibition of Xcv (FIG. 1A). This result was notstatistically significant based on analysis of variance and Tukey'shonestly significant difference (Tukey's HSD, P<0.05) in a firstanalysis, but was significant in a second analysis with the exception ofthe 1:10 ratio. A similar pattern was observed when LP:GOx ratios wereanalyzed using the 10× substrate concentration (see FIG. 1B). Inanalysis 2, the 1:1 ratio resulted in significantly larger zones ofinhibition compared to all other treatments. Ratios of 1:5 and 1:10 alsohad significantly more efficacy than the 10:1 and 5:1 ratios. The 10×substrate concentration resulted in significantly larger zones ofinhibition than the 1× substrate concentration in both analysis (seeFIG. 1C). Optimal dry enzyme and substrate disk formulae for maximizingbactericidal efficacy was determined to be a 1:1 LP:GOx molar ratio and10× substrate concentration.

Fungicidal Enzyme Concentration Optimization

Fusarium graminearum was more sensitive to the dry enzyme disks thanRhizoctonia solani (see FIG. 2). There was a pattern of increasinggrowth suppression with increasing enzyme concentration observed forboth F. graminearum and R. solani. Due to high variance amongreplicates, few of the observed differences were statisticallysignificant (Tukey's HSD, P<0.05). Rhizoctonia solani growth wasinhibited significantly more at the 10× concentration than the 1×concentration (see FIG. 2). Overall, there was a dose effect of LP:GOxenzyme concentration on fungal growth, and the 10× enzyme concentrationresulted in the greatest inhibitory effect by reducing growth of R.solani and F. graminearum by 22% and 50%, respectively.

Optimization of Activity Against Oomycetes

Three LP:GOx concentrations were analyzed in dry enzyme disks for theirefficacy against P. ultimum. The lowest concentration (0.0034×) did notinhibit P. ultimum growth relative to the control; however, the 0.68×and 2× concentrations both killed the culture plugs, effectivelyreducing growth by 100% (see FIG. 3).

Example 2—Synergy Between Chemical Fungicides and Immobilized LP:GOx

Materials and Methods

The effect of combining commercial chemical fungicide with immobilizedenzymes on microbial growth was analyzed in-vitro on two species ofoomycete plant pathogens belonging to the genus Pythium. One isolateeach of Pythium ultimum (isolate ‘Geneva 16’) and Pythium aphanidermatum(isolate ‘Pa58’) were both collected in New York State and isolated andidentified by Professor Eric Nelson (Cornell University Section of PlantPathology and Plant-Microbe Biology, Cornell University, Ithaca, N.Y.).https://pppmb.cals.cornell.edu/people/eric-nelson. Isolates were storedon corn meal agar (CMA).

A suspension of lactoperoxidase (LP) and glucose oxidase (GOx) wasprepared in a 1:1 M ratio at a concentration of 1.613 μM and pH 7.4.Enzymes were immobilized by combining the 1.613 μM enzyme suspensionwith a 1.277 mg/ml nanoparticle (NP) suspension (pH 3) in a 1:1 volumeratio. Immobilized enzymes were then combined with solid substratecomponents as described in Table 3. Separate dry substrate disks weremade according to the substrate disk compositions listed in Table 3.Liquid enzyme and substrate disk suspensions were dispensed ontoparafilm in 50 μl aliquots and dried under vacuum for approximately 1hour. Dry enzyme and substrate disks were stored at ambient temperature(approximately 22° C.).

Daconil® fungicide (chlorothalonil 26.6%, manufactured by GardenTech,Palatine, Ill., purchased from Amazon, Seattle, Wash. cat. no.B000RUGIY0).) Daconil® was mixed with sterile DDI H₂O to create a seriesof 4 dilutions (0.78% Daconil®, 0.078% Daconil®, 0.039% Daconil®,0.0078% Daconil®). The highest concentration analyzed (0.78% Daconil®)is twice the highest label rate for vegetables, and was just below thehalf maximal effective concentration (EC₅₀) value for Daconil® onPythium ultimum. The EC₅₀ is defined as the concentration of fungicidethat induces a response halfway between the baseline and maximumresponse. The EC₅₀ value for Daconil® on Pythium ultimum was 0.2% basedon regression analysis of the data shown in FIG. 5. Based on theregression it was estimated that the full fungicidal concentration isapproximately 5% Daconil®.

Fungicides were applied to the center of an 85 mm petri dish containingCMA by stacking two sterile 7 mm filter paper discs on the center ofeach plate and applying 50 μl of fungicide solution or sterile DDI H₂O(control) to the stacked discs. Treatments that includedDaconil®+immobilized enzyme disks were prepared by placing, in a stackon the center of each CMA plate, one substrate disk, one enzyme disk,and 2 filter paper discs with 50 μl fungicide applied as the last step.A 7 mm plug of actively growing Pythium mycelia was placed, mycelia sidedown, directly on top of the treatment at the center of each plate. Theentire analysis was done once for Pythium ultimum and Pythiumaphanidermatum. Treatments were as follows:

-   -   Daconil® (0.78%) only    -   Daconil® (0.078%) only    -   Daconil® (0.039%) only    -   Daconil® (0.0078%) only    -   Control—H₂O on filter paper    -   Control—1 dry enzyme disk and 1 dry substrate disk+H₂O on filter        paper    -   Daconil® (0.78%)+1 dry enzyme disk and 1 dry substrate disk    -   Daconil® (0.078%)+1 dry enzyme disk and 1 dry substrate disk    -   Daconil® (0.039%)+1 dry enzyme disk and 1 dry substrate disk    -   Daconil® (0.0078%)+1 dry enzyme disk and 1 dry substrate disk

Plates were stored on the bench and colonies allowed to grow for 2 daysbefore two perpendicular colony diameter measurements per plate wererecorded. The two perpendicular measurements were averaged for eachplate.

TABLE 3 Solid immobilized enzyme and substrate disk compositions Enzymedisk composition Substrate disk composition Component ConcentrationComponent Concentration Potassium iodide 0.3 mM Potassium 0.3 mM iodideAmmonium 0.5 mM Ammonium 0.4 mM thiocyanate thiocyanate Carboxymethyl0.7% Carboxymethyl 0.7% cellulose cellulose LP + GOx (1:1 ratio)  81 nMAvicel 2.0% Glucose  50 M

Daconil®+enzyme disk treatments resulted in increased suppression of P.ultimum and P. aphanidermatum compared to Daconil®-only and enzymedisk-only treatments (see FIGS. 4, 5, 6, and 7). The additive effect ofDaconil®+enzyme disk was calculated by adding together the reduction ingrowth due to Daconil® only and the reduction in growth due to enzymedisk only. The enzyme disk-only treatment resulted in a 46% and 84%reduction in P. ultimum and P. aphanidermatum growth, respectively. Theadditive effect of Daconil®+enzyme disk exceeded 100% for P.aphanidermatum due to its high sensitivity to the enzyme disk.Therefore, the additive and synergistic effects are only reported for P.ultimum (see FIG. 4). Synergistic activity of Daconil®+enzyme disk on P.ultimum ranged from a 13% increase in growth suppression at the highestDaconil® concentration to 32% growth suppression at the lowest Daconil®concentration. Synergism between Daconil® and enzyme disk was observedin each of the four Daconil® concentrations analyzed on P. ultimum (seeFIG. 4). Values reported in FIGS. 4 and 5 are the percent reduction inPythium growth relative to the untreated control.

In-vitro exposure of P. ultimum and P. aphanidermatum to the commercialchemical fungicide Daconil® at four concentrations, enzyme disk only,and Daconil®+enzyme disk resulted in reduced mycelial growth in everytreatment relative to the untreated control. The effect of the solidsubstrate disk only and sterile filter paper+H₂O were analyzed ascontrols and neither were found to inhibit mycelial growth. Thesynergistic effect of the combination of Daconil® and enzyme disk oninhibition of P. ultimum was observed at all four Daconil®concentrations used (see FIG. 4). This showed that Daconil®, orchlorothalonil-containing products, had enhanced efficacy through theinclusion of immobilized enzyme disk. Additionally, less chemicalfungicide may be used in the presence of enzyme disks to achieve thesame level of fungal suppression or disease control as higher fungicideapplication rates in the absence of enzyme disks.

Synergism between Daconil® and enzyme disks could not be calculated forP. aphanidermatum due to the high sensitivity of this species to theenzyme disk used in this analysis. However, treatments ofDaconil®+enzyme disk consistently resulted in greater suppression of P.aphanidermatum growth compared to treatments of Daconil® only and enzymedisk only (see FIG. 5). The effect of lower enzyme concentrations ofLP:GOx in immobilized enzyme disks with and without Daconil® on P.aphanidermatum may also be analyzed.

Example 3—Synergy Between Antibiotics and Immobilized LP:GOx

Materials and Methods

Antibiotic suspensions (ampicillin) were diluted to four concentrationsso that the final amounts applied to sterile filter paper disks were 0.1μg, 1 μg, 10 μg and 100 μg. The minimum inhibitory concentration (MIC)of ampicillin for Xanthomonas campestris was estimated to be between 10μg and 50 μg. The MIC is defined as the lowest concentration ofantibiotic that completely inhibits growth of the bacteria beingevaluated and the minimum bactericidal concentration (MBC) is defined asthe lowest concentration of antibiotic at which bacteria are killed.This was not determined for Xanthomonas campestris because resistantcolonies persisted at the highest antibiotic concentration. Dry enzymedisks were made according to the formula in Table 3 with the exceptionof the enzyme concentration which is 238 nM. 10× dry substrate diskswere made according to the formula in Table 2. The following treatmentswere analyzed:

-   -   0.1 μg antibiotic only    -   1 μg antibiotic only    -   10 μg antibiotic only    -   100 μg antibiotic only    -   Control—sterile DDI H₂O on filter paper    -   Control—1 dry enzyme disk and 1 dry substrate disk+sterile DDI        H₂O on filter paper    -   Control—1 dry substrate disk+sterile DDI H₂O on filter paper    -   0.1 μg antibiotic+1 dry enzyme disk and 1 dry substrate disk    -   1 μg antibiotic only+1 dry enzyme disk and 1 dry substrate disk    -   10 μg antibiotic only+1 dry enzyme disk and 1 dry substrate disk    -   100 μg antibiotic only+1 dry enzyme disk and 1 dry substrate        disk

The analysis was performed by creating bacterial lawns of Xcv on 85-mmpetri dishes as described in Example 1. It followed the methodsdescribed in Example 2 with the exception of using antibioticsuspensions at four concentrations instead of fungicides. It alsomeasured zones of inhibition of bacterial growth after two days ratherthan placing 7-mm fungal plugs on top of the treatments and measuringcolony diameters.

Results:

Of the four ampicillin-alone treatments, only the 100 μgampicillin-alone treatment inhibited the growth of Xcv. This treatmentresulted in a ZOI of 36 mm (FIG. 8). 100 μg ampicillin-alone did notresult in a ZOC and ampicillin-resistant bacterial colonies could beseen throughout the 36 mm ZOI (FIG. 8). All four of the ampicillin+dryenzyme disk treatments resulted in ZOC ranging from 31 mm to 33.5 mm(Table 4). The 100 μg ampicillin+dry enzyme disk treatment resulted in aZOC of 32.5 mm with an additional ZOI extending beyond the boundary ofthe ZOC by an additional 4.5 mm. The 100 μg ampicillin+dry enzyme disktreatment resulted in a greater inhibitory effect against Xcv thaneither ampicillin alone or enzyme disk alone by clearing all viablebacteria in the ZOC surrounding the treatment and by inhibitingbacterial growth beyond the ZOC. The controls without enzyme did notinhibit growth of Xcv.

TABLE 4 Zones of clearance (ZOC) and zones of inhibition (ZOI) of Xcvsurrounding ampicillin-alone and ampicillin + dry enzyme disktreatments. ZOC ZOI Treatment (mm) (mm) 100 ug ampicillin 0 36 10 ugampicillin 0 0 1 ug ampicillin 0 0 0.1 ug ampicillin 0 0 Control-fp +H2O 0 0 100 ug ampicillin + enzyme 32.5 37 disk 10 ug ampicillin +enzyme disk 33.5 0 1 ug ampicillin + enzyme disk 31 0 0.1 ugampicillin + enzyme 32.5 0 disk Control - substrate disk + fp + 0 0 H2OControl - enzyme disk 33 0

Example 4—Plant Pathogen Control Using Stabilized Biocidal Enzymes andBiocidal Vegetable Extracts

Essential oils, such as tea tree oil, have antimicrobial properties. Teatree oil (TTO) in combination with stabilized biocidal enzymes,controlled plant pathogens as shown using the plant pathogenic oomycetePythium ultimum. Dilutions of TTO were impregnated into filter paper(FP) disks, combined with stabilized biocidal enzyme disks, and placedonto cornmeal agar plates with a plug of P. ultimum culture. Thereduction in colony growth was measured and compared to colony growth inthe absence of biocidal enzymes and TTO.

Materials and Methods

Stabilized biocidal enzyme disk preparation. Dry enzyme disks were madeby combining 3 μl KI (1M), 5 μl NH₄SCN (1M), 175 μl 4% carboxymethylcellulose (CMC), 295 μl stabilized lactoperoxidase+glucose oxidase(LPO+GOx) (119 nM+152.2 nM), and 3 μl blue food dye brought up to afinal volume of 1 ml with DDI H₂O. Enzyme stabilization was performed asfollows: lactoperoxidase (LPO) (125 μg/ml, pH 7.4) and glucose oxidase(GOx) (330 μg/ml, pH 7.4) were mixed to achieve a 1:1.3 M LPO:GOx ratioand stored on ice. Magnetite nanoparticles (NP) (1.277 mg/ml, pH 3,approximately 5 ml stock) were ultrasonicated at 40% amplitude for 1min, cooled to ambient temperature (approximately 21° C.) in a waterbath, and pipette mixed with the LPO:GOx enzyme suspension in a 1:1enzyme:NP ratio. Dry substrate disks were made my combining 30 μl KI(1M), 50 μl NH₄SCN (1M), 350 μl 4% CMC, 500 μl glucose (1M), and 3 μlred food dye brought up to a final volume of 1 ml with DDI H2O. Dyeswere included to differentiate enzyme disks from the substrate disks andwere not biologically active or structural components of the disks. Eachsolution was pipette mixed several times and vortexed briefly. Solutionswere dispensed in 50 μl aliquots onto parafilm and dried at ambienttemperature in a vacuum oven containing desiccant at −50 kPa. Afterapproximately 2 hours, dry enzyme and substrate disks were stored in thedark at 4° C. until use.

Tea tree oil disk preparation and culture growth assay. Tea tree oil(Active Ingredient (AI): tea tree oil 100%, Mason Natural, Miami Lakes,Fla.) (TTO) was diluted in sterile DDI H₂O. Whatman® qualitative grade 1filter paper (Sigma-Aldrich) was cut into 7 mm disks using a 3-holepunch, and disks were autoclaved prior to use. FP disks were impregnatedwith 5 μl of each of four TTO dilutions. Control FP disks wereimpregnated with 5 μl sterile DDI H₂O. TTO and enzyme disk interactionswere tested by placing one substrate disk on the center of a petri dishcontaining corn meal agar, followed by one enzyme disk, oneTTO-impregnated FP disk, and one culture plug mycelia-side down. Finalenzyme concentrations in enzyme disks were 4 nM for P. ultimum. Cultureplugs of P. ultimum measured 7 mm in diameter. Each experiment includedthe same TTO dilution series plated without substrate and enzyme disks,as well as a substrate+enzyme disk-only treatment and a non-treatedcontrol. All plates, including controls, contained a FP disk, and eachtreatment was replicated once. Plates were left on the bench at ambienttemperature for 2 days. At that time, control colonies had nearly grownto the plate edge. Two perpendicular colony diameter measurements wererecorded for P. ultimum.

Results

Tea tree oil combined with the stabilized enzyme formulation resulted ina statistically synergistic effect at the two lowest TTO concentrationsand was additive at the two highest concentrations (Table 5). Thecombined effects were significantly greater than the effects of theenzyme formulation alone and three of the four TTO concentrations alone.The highest TTO concentration alone produced a significantly greatereffect than enzyme formulation alone, and was not significantlydifferent from any of the four combined effects (Table 5).

TABLE 5 Inhibition of Pythium ultimum by the stabilized LPO formulationalone and in combination with tea tree oil using TTO-impregnated filterpaper disks. Combined Tea Reduction in growth^(a) effect tree oil (+)(−) Tukey's (observed − Combination dose enzyme SD enzyme SD HSDexpected)^(b) result 30% 100% a^(c ) 0.0% 91% a 12.1% 23.4%  0% additive20% 100% a  0.0% 66% b 9.4%  +7% additive 15% 100% a  0.0%  45% bc 2.1%+28% synergistic 10% 94% a 6.6% 31% c 1.6% +36% synergistic  0% 27% c4.7% NA NA ^(a)Reduction in growth relative to non-treated controls.Each value is the mean of two replicates. ^(b)Difference betweenobserved effect of fungicide (+) enzyme formulation and expectedadditive effect. Expected value calculated by adding the effect offungicide alone and the effect of the enzyme formulation alone for eachfungicide concentration. ^(c)Means followed by the same letter are notsignificantly different, Tukey's HSD (P < 0.0

Example 5—Pathogenic Nematode Control Using Biocidal Stabilized Enzymesand Stabilized Emulsified Tea Tree Oil

Root-knot nematodes from the genus Meloidogyne infect a wide array ofplants, including woody crops and vegetables, causing yield loss. Thecyst nematode Heterodera schachtii causes growth retardation in infectedplants and can cause massive yield loss at high population densities.Control of nematode populations is vital to reducing losses associatedwith reduced crop yields. Stabilized biocidal enzymes combined with TTOcontrol plant pathogens but TTO is not miscible in water and difficultto combine with biocidal enzymes. In this example, microencapsulated TTOwas combined with stabilized biocidal enzymes for the control of plantpathogens. Nematode juveniles, eggs, and cysts were incubated withtreatments containing biocidal stabilized enzymes, TTO, or both, tomeasure plant pathogenic nematodes killing or control.

Materials and Methods

Preparation of microencapsulated TTO and stabilized biocidal enzymes.Stabilized microencapsulated TTO (40%, 40× strength) was prepared bycombining 2 ml of a 2% (w/w) solution of EHM (Ethyl hydroxyethylcellulose EHM300, Akzonobel), 4 ml TTO (pure), and 4 ml DDI H₂O. Thissolution was sonicated two times for 1 minute each at 40% amplitude, ¼thinch horn. The 20×TTO substrate solution was made by combining 100 μlstabilized EHM TTO mix (40% TTO), 500 μl glucose (1M), 3 μl red fooddye, and brought up to a final volume of 1 ml with DDI H₂O. The standardsubstrate solution was made by combining 500 μl glucose (1M), 3 μl redfood coloring dye, and brought up to a final volume of 1 ml with DDIH₂O. The enzyme solution was made by combining 3 μl KI (1M), 5 μl NH₄SCN(1M), 70 μl 4% carboxymethyl cellulose (CMC), 295 μl stabilizedlactoperoxidase+glucose oxidase (LPO+GOx) (119 nM+152.2 nM), and 3 μlblue food dye brought up to a final volume of 1 ml with DDI H₂O. Enzymestabilization was performed as follows: LPO (125 μg/ml, pH 7.4) and GOx(330 μg/ml, pH 7.4) were mixed to achieve a 1:1.3 M LPO:GOx ratio andstored on ice. Magnetite nanoparticles (NP) (1.277 mg/ml, pH 3,approximately 5 ml stock) were ultrasonicated at 40% amplitude for 1min, cooled to ambient temperature (approximately 21° C.) in a waterbath, and pipette mixed with the LPO:GOx enzyme suspension in a 1:1enzyme:NP ratio. Each solution was pipette mixed several times andvortexed briefly. Solutions were stored in the dark at 4° C. until use.

Control of plant pathogenic nematodes using microencapsulated TTO andstabilized biocidal enzymes. For the nematode juvenile analyses, a stocksuspension of Meloidogyne incognita juveniles was prepared at aconcentration of 300 juveniles/ml. Four treatment solutions wereprepared as follows (1 ml total volume): Treatment solution (Tmt) 1=33μl enzyme solution (1.96 nM lactoperoxidase+2.51 nM glucose oxidase)+33μl standard substrate solution (500 mM glucose)+934 μl water, Tmt 2=33μl 0.066% tea tree oil (TTO) solution+967 μl water, Tmt 3=33 μl enzymesolution (1.96 nM lactoperoxidase+2.51 nM glucose oxidase)+33 μl 0.066%TTO solution+934 μl water, and Tmt 4 (control)=1000 μl water. Eachtreatment solution was combined with 1 ml of the M. incognita stocksuspension in a 10 ml beaker for 300 juveniles per experiment. Analyseswere triplicated for each treatment resulting in 12 samples in total.They were stored individually in tightly sealed boxes to reduce volumeloss due to evaporation at room temperature for three days. After threedays, 100 juveniles were removed from each sample and counted as live ordead. If movement was observed during the count, the juvenile wascounted as alive. If no movement was observed, the juvenile was countedas dead.

In the nematode cyst analyses, each sample (10 ml beaker) receivedapproximately 50 Heterodera schachtii cysts. Cysts were hand-pickedunder a dissecting microscope and added to each beaker. Four treatmentsolutions were prepared as follows (2 ml total volume): Tmt1=33 μlenzyme solution (1.96 nM lactoperoxidase+2.51 nM glucose oxidase)+33 μlstandard substrate solution (500 mM glucose)+1934 μl water, Tmt2=33 μl0.066% tea tree oil (TTO) solution+1967 μl water, Tmt3=33 μl enzymesolution (1.96 nM lactoperoxidase+2.51 nM glucose oxidase)+33 μl 0.066%TTO solution+1934 μl water, and Tmt4 (control)=2000 μl water. Eachsample with 50 cysts received 2 ml of treatment solution (2 ml totalexperimental volume). Analyses were triplicated for each treatmentresulting in 12 samples in total. Samples were stored individually inboxes at room temperature for 1 week. After 1 week, 300 μl of the samplevolume was removed and the number of live or dead juveniles was countedas previously described.

In the nematode egg analyses, a stock suspension of H. schachtii eggswas prepared at a concentration of 1200 eggs/ml. Each sample (10 mlbeaker) received 800 μl of the egg suspension for 960 eggs perexperimental unit. Four treatment solutions were prepared as follows(1.2 ml total volume): Tmt1=33 μl enzyme solution (1.96 nMlactoperoxidase+2.51 nM glucose oxidase)+33 μl standard substratesolution (500 mM glucose)+1134 μl water, Tmt2=33 μl 0.066% tea tree oil(TTO) solution+1167 μl water, Tmt3=33 μl enzyme solution (1.96 nMlactoperoxidase+2.51 nM glucose oxidase)+33 μl 0.066% TTO solution+1134μl water, and Tmt4 (control)=1200 μl water. Each sample with 800 μl H.schachtii egg suspension received 1.2 ml of treatment solution (2 mltotal experimental volume). Analyses were triplicated for each treatmentresulting in 12 samples in total. Samples were stored individually inboxes at room temperature for 1 week. After 1 week, 300 μl of the samplevolume was removed and the number of live or dead juveniles was countedas previously described.

Results

Results from each of the analyses showed effective control of nematodesby stabilized enzymes. FIG. 9 shows the result of the nematode juvenileexperiment for which the treatments with stabilized enzymes alone andstabilized enzymes with tea tree oil showed 100% effectiveness atkilling M. incognita juveniles. 78% of the nematode juveniles werekilled in a solution of tea tree oil alone. Only 5% of nematodejuveniles were killed in the control sample.

FIGS. 10A and 10B show the results of the nematode egg and cystanalyses. In FIG. 10A, the total number of nematode juveniles hatchedfrom H. schachtii cysts, both live and dead, observed in the treatmentswith stabilized enzymes, TTO, or both is greatly reduced from the totalnumber of nematodes observed in the control solution. The treatmentsinhibit hatching from the cysts. The enzyme treatment is more inhibitoryto hatching than TTO alone. Juveniles that do hatch from cysts followingtreatment are nearly all dead by the end of the incubation period. FIG.10B shows the number of nematode juveniles hatched from eggs, both liveand dead, observed in the treatments with stabilized enzymes, TTO, orboth. In this case, the treatments do not inhibit hatching, though thejuveniles that do hatch are subsequently killed by the treatments duringthe incubation period.

Example 6—Plant Pathogen Control Using Stabilized Biocidal Enzymes andMancozeb

Mancozeb is a non-systemic fungicide used for agriculture to controlfungal diseases in a wide array of field crops. An enhanced fungicidaleffect from the combination of mancozeb and stabilized biocidal enzymeswas shown. Solid culture growth media was amended with mancozeb at fourconcentrations. Stabilized biocidal enzyme disks and standard substratedisks were placed onto these plates with a plug of P. ultimum or F.graminearum culture. The reduction in colony growth was measured andcompared to colony growth in the absence of biocidal enzymes andmancozeb.

Materials and Methods

Stabilized biocidal enzyme disk preparation. Dry enzyme disks were madeby combining 3 μl KI (1M), 5 μl NH4SCN (1M), 175 μl 4% carboxymethylcellulose (CMC), 295 μl stabilized lactoperoxidase+glucose oxidase(LPO+GOx) (119 nM+152.2 nM), and 3 μl blue food dye brought up to afinal volume of 1 ml with DDI H₂O. Enzyme stabilization was performed asfollows: lactoperoxidase (LPO) (125 μg/ml, pH 7.4) and glucose oxidase(GOx) (330 μg/ml, pH 7.4) were mixed to achieve a 1:1.3 M LPO:GOx ratioand stored on ice. Magnetite nanoparticles (NP) (1.277 mg/ml, pH 3,approximately 5 ml stock) were ultrasonicated at 40% amplitude for 1min, cooled to ambient temperature (approximately 21° C.) in a waterbath, and pipette mixed with the LPO:GOx enzyme suspension in a 1:1enzyme:NP ratio. Dry substrate disks were made my combining 30 μl KI(1M), 50 μl NH₄SCN (1M), 350 μl 4% CMC, 500 μl glucose (1M), and 3 μlred food dye brought up to a final volume of 1 ml with DDI H₂O. Dyeswere included to differentiate enzyme disks from the substrate disks andwere not biologically active or structural components of the disks. Eachsolution was pipette mixed several times and vortexed briefly. Solutionswere dispensed in 50 μl aliquots onto parafilm and dried at ambienttemperature in a vacuum oven containing desiccant at −50 kPa. Afterapproximately 2 hours, dry enzyme and substrate disks were stored in thedark at 4° C. until use.

Mancozeb amended plates and culture growth assays. Corn meal agar (CMA)and potato dextrose agar (PDA) (for P. ultimum and F. graminearum,respectively) were amended with mancozeb flowable with zinc (Activeingredient (AI): mancozeb 37%, Bonide, Oriskany, N.Y.) to achieve finalconcentrations of 10 mg/l, 5 mg/l, 2 mg/l, 0.5 mg/l, and 0 mg/l(controls). Mancozeb and enzyme disk interactions were analyzed byplacing one substrate disk on the center of each petri dish containingPDA amended with mancozeb (F. graminearum) or CMA amended with mancozeb(P. ultimum). These were followed by one enzyme disk and one cultureplug mycelia-side down. Final enzyme concentrations in enzyme disks were4 nM for P. ultimum and 119 nM for F. graminearum and based on resultsfrom preliminary enzyme formula optimization experiments. Plugs of P.ultimum measured 7 mm in diameter and plugs of F. graminearum measured 4mm in diameter.

Preliminary testing revealed that P. ultimum is more sensitive to theenzyme treatment than F. graminearum. Thus, enzyme concentrations andplug sizes were chosen to achieve a measurable growth reduction, withoutbeing completely inhibitive, in enzyme-only treatments compared tonon-treated controls. Each analysis included the same fungicide dilutionseries plated without substrate and enzyme disks as well as asubstrate+enzyme disk-only treatment and a non-treated control. Eachtreatment was replicated once. Plates were left on the bench at ambienttemperature for 2 days for P. ultimum, and 5 days for F. graminearum.Following the incubation period, control colonies had nearly grown tothe plate edge. Two perpendicular colony diameter measurements wererecorded for P. ultimum. Colonies of F. graminearum measured using thepublic domain image processing program ImageJ.

Results

Mancozeb combined with the stabilized enzyme formulation resulted in astatistically synergistic effect on P. ultimum at the lowest fungicideconcentration and was additive at the three highest concentrations(Table 6). The combined effects were significantly greater than theeffects of the enzyme formulation alone and all four fungicideconcentrations alone. The effect of the enzyme formulation alone wassignificantly greater than the three lowest fungicide concentrationsalone, but was not significantly different from the highestconcentration alone (Table 6). Combined activity against F. graminearumwas additive at all four fungicide concentrations (Table 6). Thecombined effects were significantly greater than the correspondingfungicide concentrations alone at the three highest concentrations. Theeffect of the enzyme formulation alone was not significantly differentfrom any of the combined effects nor the fungicides alone (Table 6).

TABLE 6 Inhibition of Pythium ultimum and Fusarium graminearum by thestabilized LPO formulation alone and in combination with mancozeb usingfungicide-amended media. Combined Mancozeb Reduction in P. ultimumgrowth^(a) effect concentration (+) (−) Tukey's (observed − Combination(mg/l) enzyme SD enzyme SD HSD expected)^(b) result 10  53% a^(c) 0.3%23% d 1.7% 6.04% +4% additive 5 45% b 0.5% 16% e 0.4% +3% additive 2 34%c 1.3% 10% e 0.8% −2% additive 0.5 35% c 3.7%  2% f 0.5% +7% synergistic0 26% d 0.9% NA NA Combined Mancozeb Reduction in F. graminearumgrowth^(a) effect concentration (+) (−) Tukey's (observed − Combination(mg/l) enzyme SD enzyme SD HSD expected)^(b) result 10 58% a^(c ) 5.7%26% bcd 6.0% 32.4% +3% additive 5 53% ab 15.6% 17% cd  5.4% +7% additive2 51% ab 5.3% 1% d  5.1% +21%  additive 0.5  39% abc 10.9% 6% cd 3.8%+4% additive 0  29% abcd 8.9% NA NA ^(a)Reduction in growth relative tonon-treated controls. Each value is the mean of two replicates.^(b)Difference between observed effect of fungicide (+) enzymeformulation and expected additive effect. Expected value calculated byadding the effect of fungicide alone and the effect of the enzymeformulation alone for each fungicide concentration. ^(c)Means followedby the same letter are not significantly different, Tukey's HSD (P <0.05).

All publications and patent documents disclosed or referred to hereinare incorporated by reference in their entirety. The foregoingdescription has been presented only for purposes of illustration anddescription. This description is not intended to limit the invention tothe precise form disclosed. It is intended that the scope of theinvention be defined by the claims appended hereto.

What is claimed:
 1. A solid fungicidal composition, comprising; a. afirst component having self-assembled mesoporous aggregates of magneticnanoparticles comprising a hydrogen peroxide producing enzyme and a freeradical producing enzyme; b. a second component having a first substratefor said hydrogen peroxide producing enzyme and a second substrate forsaid free radical producing enzyme; and c. a chemical fungicide; whereinsaid composition is essentially inactive, wherein exposure of said firstand second components to hydration or oxygen activates said compositionand results in said substrate for said hydrogen peroxide producingenzyme being oxidized into hydrogen peroxide, wherein said hydrogenperoxide acts as a substrate for said free radical producing enzyme, andwherein said free radicals are produced having fungicidal activities. 2.The fungicidal composition of claim 1, wherein said chemical fungicideis selected from the group consisting of mefenoxam, myclobutanil,chlorothalonil, prothioconazole, trifloxystrobin, propiconazole,mancozeb, and copper.
 3. The fungicidal composition of claim 2, whereinsaid chemical fungicide is chlorothalonil.
 4. The fungicidal compositionof claim 2, wherein said chemical fungicide is mancozeb.
 5. A solidbactericidal composition, comprising; a. a first component havingself-assembled mesoporous aggregates of magnetic nanoparticlescomprising a hydrogen peroxide producing enzyme and a free radicalproducing enzyme; b. a second component having a first substrate forsaid hydrogen peroxide producing enzyme and a second substrate for saidfree radical producing enzyme; and c. a chemical antibiotic; whereinsaid composition is essentially inactive, wherein exposure of said firstand second components to hydration or oxygen activates said compositionand results in said substrate for said hydrogen peroxide producingenzyme being oxidized into hydrogen peroxide, wherein said hydrogenperoxide acts as a substrate for said free radical producing enzyme, andwherein said free radicals are produced having bactericidal activities.6. The bactericidal composition of claim 5, wherein said chemicalantibiotic is selected from the group consisting of ampicillin,streptomycin, vancomycin, and copper.
 7. A liquid fungicidalcomposition, comprising; a. a first component having self-assembledmesoporous aggregates of magnetic nanoparticles comprising a freeradical producing enzyme; b. a second component having a substrate forsaid free radical producing enzyme and a hydrogen peroxide source; andc. a chemical fungicide; wherein said composition is essentiallyinactive, wherein mixing said first and second components activates saidcomposition and results in said hydrogen peroxide source acting as asubstrate for said free radical producing enzyme, and wherein said freeradicals are produced having fungicidal activities.
 8. A liquidbactericidal composition, comprising; a. a first component havingself-assembled mesoporous aggregates of magnetic nanoparticlescomprising a free radical producing enzyme; b. a second component havinga substrate for said free radical producing enzyme and a hydrogenperoxide source; and c. a chemical antibiotic; wherein said compositionis essentially inactive, wherein mixing said first and second componentsactivates said composition and results in said hydrogen peroxide sourceacting as a substrate for said free radical producing enzyme, andwherein said free radicals are produced having bactericidal activities.9. The fungicidal composition of claim 1, wherein the final chemicalfungicide concentration is between about 10% and 2500% of the halfmaximal effective concentration (EC₅₀).
 10. The bactericidal compositionof claim 5, wherein the final chemical antibiotic concentration isbetween about 1 and 100% of the minimum inhibitory concentration (MIC)or minimum bactericidal concentration (MBC).
 11. The fungicidalcomposition of claim 1, further comprising a chemical antibiotic. 12.The fungicidal composition of claim 1, wherein said mesoporousaggregates of magnetic nanoparticles have an iron oxide composition. 13.The fungicidal composition of claim 1, wherein said mesoporousaggregates of magnetic nanoparticles have a magnetic nanoparticle sizedistribution in which at least 90% of magnetic nanoparticles have a sizeof at least about 3 nm and up to about 30 nm, and an aggregated particlesize distribution in which at least about 90% of said mesoporousaggregates of magnetic nanoparticles have a size of at least about 10 nmand up to 500 nm.
 14. The fungicidal composition of claim 1, whereinsaid mesoporous aggregates of magnetic nanoparticles possess a saturatedmagnetization of at least 10 emu/g.
 15. The fungicidal composition ofclaim 1, wherein said free-radical-producing enzyme and hydrogenperoxide producing enzyme are contained in said mesoporous aggregates ofmagnetic nanoparticles in up to about 100% of saturation capacity. 16.The fungicidal composition of claim 1, wherein said hydrogen peroxidegenerating enzyme is an oxidase.
 17. The fungicidal composition of claim1, wherein said oxidase is glucose oxidase or alcohol oxidase.
 18. Anagricultural product, comprising the fungicidal composition of claim 1.19. A liquid pesticide product comprising the fungicidal composition ofclaim
 7. 20. A seed coating, comprising the fungicidal composition ofclaim
 1. 21. A seed, comprising the seed coating of claim 20, whereinsaid seed is selected from the group consisting of vegetable, fruit,flower and field crop.
 22. The seed of claim 21, wherein said vegetableseed is selected from the group consisting of tomato, pea, onion,garlic, parsley, oregano, basil, cilantro, carrot, cabbage, corn,cucumber, radish, pepper, broccoli, cauliflower, cucumber, spinach,kale, chard, artichoke, and lettuce.
 23. The seed of claim 21, whereinsaid fruit seed is selected from the group consisting of citrus, tomato,orange, lemon, lime, avocado, clementine, apple, persimmon, pear, peach,nectarine, berry, strawberry, raspberry, grape, blueberry, blackberry,cherry, apricot, gourds, squash, zucchini, eggplant, pumpkin, coconut,guava, mango, papaya, melon, honeydew, cantaloupe, watermelon, banana,plantain, pineapple, quince, sorbus, loquata, plum, currant,pomegranate, fig, olive, fruit pit, a nut, peanut, almond, cashew,hazelnut, brazil nut, pistachio, and macadamia.
 24. The seed of claim21, wherein said field crop is selected from the group consisting ofcorn, wheat, soybean, canola, sorghum, potato, sweet potato, yam,lentils, beans, cassava, coffee, hay, buckwheat, oat, barley, rape,switchgrass, elephant grass, beet, sugarcane, and rice.
 25. The seed ofclaim 21, wherein said flower seed is selected from the group consistingof annual, perennial, bulb, flowering woody stem, carnation, rose,tulip, poppy, snapdragon, lily, mum, iris, alstroemeria, pom, fuji, andbird of paradise.
 26. An animal bedding, comprising the fungicidalcomposition of claim
 1. 27. A wound dressing, comprising the fungicidalcomposition of claim
 1. 28. A fabric, comprising the fungicidalcomposition of claim
 1. 29. A method of improving a plant product yield,comprising exposing the seed of claim 21 to hydration and oxygenationprior to or during the planting or germination of said plant.
 30. Amethod of improving an animal product yield, comprising exposing theanimal bedding of claim 26 to hydration and oxygen prior to or duringuse by said animal.
 31. The method of claim 30, wherein said hydrationis from said animal's urine.
 32. The method of claim 30, wherein saidanimal product is selected from the group consisting of live animals,milk, meat, fat, eggs, bodily fluids, blood, serum, antibodies, enzymes,rennet, bone, animal byproducts, and animal waste.
 33. The method ofclaim 30, wherein said animal is selected from the group consisting ofcows, pigs, chickens, turkeys, horses, sheep, goats, donkeys, mules,ducks, geese, buffalo, camels, yaks, llama, alpacas, mice, rats, dogs,cats, hamsters, guinea pigs, reptiles, amphibians, parrots, parakeets,cockatiels, canaries, pigeons, doves, and insects.
 34. A method ofreducing sepsis, comprising administering the wound dressing of claim 27to a wound.
 35. A method of producing the fungicidal composition ofclaim 1, comprising formulating said first component with a matrixmaterial selected from the group consisting of water-soluble cellulosederivatives, water-solvatable cellulose derivatives, alginatederivatives, and chitosan derivatives and formulating said secondcomponent with a matrix material selected from the group consisting ofwater-soluble cellulose derivatives, water-solvatable cellulosederivatives, alginate derivatives, and chitosan derivatives.
 36. Themethod of claim 35, wherein said first component is further subjected tospray drying, freeze drying, drum drying, pulse combustion drying, orrotary seed coating.
 37. The method of claim 35, wherein said secondcomponent is further subjected to spray drying, freeze drying, drumdrying, pulse combustion drying, or rotary seed coating.
 38. A method ofreducing or eliminating fungal growth, comprising spraying a substancewith the liquid fungicidal composition of claim
 7. 39. A method ofprotecting an agricultural product from a pathogen, comprising exposingsaid product to the fungicidal composition of claim
 7. 40. The method ofclaim 39, wherein said pathogen is a plant, animal, or human pathogen.41. The method of claim 39, wherein said pathogen is a fungus or anoomycete.
 42. The method of claim 41, wherein said fungus is selectedfrom the group consisting of Rhizoctonia species and Fusarium species.43. The method of claim 41, wherein said pathogen is a bacteriumselected from the group consisting of Xanthomonas campestris,Clavibacter michiganensis, Acidovorax avenae, Pseudomonas viridiflava,Pseudomonas syringae, Escherichia coli, Salmonella species, and Listeriaspecies.
 44. The method of claim 41, wherein said oomycete is selectedfrom the group consisting of Pythium species and Phytophthora species.45. A method of reducing or eliminating damping off in a plant,comprising exposing said plant to the bactericidal composition of claim5.
 46. A solid nematocidal composition, comprising; a. a first componenthaving self-assembled mesoporous aggregates of magnetic nanoparticlescomprising a hydrogen peroxide producing enzyme and a free radicalproducing enzyme; and b. a second component having a first substrate forsaid hydrogen peroxide producing enzyme and a second substrate for saidfree radical producing enzyme; wherein said composition is essentiallyinactive, wherein exposure of said first and second components tohydration or oxygen activates said composition and results in saidsubstrate for said hydrogen peroxide producing enzyme being oxidizedinto hydrogen peroxide, wherein said hydrogen peroxide acts as asubstrate for said free radical producing enzyme, and wherein said freeradicals are produced having fungicidal activities.
 47. A liquidnematocidal composition, comprising; a. a first component havingself-assembled mesoporous aggregates of magnetic nanoparticlescomprising a free radical producing enzyme; and b. a second componenthaving a substrate for said free radical producing enzyme and a hydrogenperoxide source; wherein said composition is essentially inactive,wherein mixing said first and second components activates saidcomposition and results in said hydrogen peroxide source acting as asubstrate for said free radical producing enzyme, and wherein said freeradicals are produced having nematocidal activities.
 48. The nematocidalcompositions of claim 46, further comprising an essential oil.
 49. Thenematocidal compositions of claim 48, wherein said essential oil isselected from the group consisting of tea tree (TTO), aegle, ageratum,citrus, citronella, orange, pine, eucalyptus, marigold, geranium,lemongrass, orange, palmarosa, mint, peppermint, cinnamon, clove,rosemary, thyme, garlic, oregano, anise, cumin, turmeric, curcuma,caraway, fennel, onion, and patchouli oil.
 50. The nematocidalcompositions of claim 48, wherein said essential oil is TTO.
 51. Anagricultural product, comprising the nematocidal composition of claim46.
 52. A liquid pesticide product, comprising the nematocidalcomposition of claim
 47. 53. A seed coating, comprising the nematocidalcomposition of claim
 46. 54. The seed coating of claim 53, wherein saidseed is selected from the group consisting of vegetable, fruit, flowerand field crop.
 55. The seed coating of claim 54, wherein said vegetableseed is selected from the group consisting of tomato, pea, onion,garlic, parsley, oregano, basil, cilantro, carrot, cabbage, corn,cucumber, radish, pepper, broccoli, cauliflower, cucumber, spinach,kale, chard, artichoke, and lettuce.
 56. The seed coating of claim 54,wherein said fruit seed is selected from the group consisting of citrus,tomato, orange, lemon, lime, avocado, clementine, apple, persimmon,pear, peach, nectarine, berry, strawberry, raspberry, grape, blueberry,blackberry, cherry, apricot, gourds, squash, zucchini, eggplant,pumpkin, coconut, guava, mango, papaya, melon, honeydew, cantaloupe,watermelon, banana, plantain, pineapple, quince, sorbus, loquata, plum,currant, pomegranate, fig, olive, fruit pit, a nut, peanut, almond,cashew, hazelnut, brazil nut, pistachio, and macadamia.
 57. The seedcoating of claim 54, wherein said field crop is selected from the groupconsisting of corn, wheat, soybean, canola, sorghum, potato, sweetpotato, yam, lentils, beans, cassava, coffee, hay, buckwheat, oat,barley, rape, switchgrass, elephant grass, beet, sugarcane, and rice.58. The seed coating of claim 54, wherein said flower seed is selectedfrom the group consisting of annual, perennial, bulb, flowering woodystem, carnation, rose, tulip, poppy, snapdragon, lily, mum, iris,alstroemeria, pom, fuji, and bird of paradise.
 59. An animal bedding,comprising the nematocidal composition of claim
 46. 60. A method ofimproving a plant product yield, comprising exposing the seed coating ofclaim 53 to hydration and oxygenation prior to or during the planting orgermination of said plant.
 61. A method of improving an animal productyield, comprising exposing the animal bedding of claim 59 to hydrationand oxygen prior to or during use by said animal.
 62. The method ofclaim 61, wherein said hydration is from said animal's urine.
 63. Themethod of claim 61, wherein said animal product is selected from thegroup consisting of live animals, milk, meat, fat, eggs, bodily fluids,blood, serum, antibodies, enzymes, rennet, bone, animal byproducts, andanimal waste.
 64. The method of claim 61, wherein said animal isselected from the group consisting of cows, pigs, chickens, turkeys,horses, sheep, goats, donkeys, mules, ducks, geese, buffalo, camels,yaks, llama, alpacas, mice, rats, dogs, cats, hamsters, guinea pigs,reptiles, amphibians, parrots, parakeets, cockatiels, canaries, pigeons,doves, and insects.
 65. A method of producing the nematocidalcomposition of claim 46, comprising formulating said first componentwith a matrix material selected from the group consisting ofwater-soluble cellulose derivatives, water-solvatable cellulosederivatives, alginate derivatives, and chitosan derivatives andformulating said second component with a matrix material selected fromthe group consisting of water-soluble cellulose derivatives,water-solvatable cellulose derivatives, alginate derivatives, andchitosan derivatives.
 66. The method of claim 65, wherein said firstcomponent is further subjected to spray drying, freeze drying, drumdrying, pulse combustion drying, or rotary seed coating.
 67. The methodof claim 65, wherein said second component is further subjected to spraydrying, freeze drying, drum drying, pulse combustion drying, or rotaryseed coating.
 68. A method of reducing or eliminating nematode growth,comprising spraying a substance with the liquid nematocidal compositionof claim
 47. 69. A method of protecting an agricultural product from anematode, comprising exposing said product to the nematocidalcomposition of claim
 47. 70. The method of claim 69, wherein saidnematode is selected from the group consisting of Meloidogyne species(spp.), Heterodera spp., Globodera spp., Pratylenchus spp.,Helicotylenchus spp., Radopholus similis, Ditylenchus dipsaci,Rotylenchulus reniformis, Xiphinema spp, Aphelenchoides spp., Toxocaraspp., Bursaphelenchus xylophilus, and trichinella spiralis.
 71. Themethod of claim 69, wherein said nematode is a Meloidogyne spp.
 72. Themethod of claim 69, wherein said nematode is trichinella spiralis. 73.The method of claim 69, wherein said nematode is a Toxocara spp.